HYBRIDIZATION PROBES AND METHODS

FIELD OF THE INVENTION

The present invention relates to compositions and methods for detection, analysis, and treatment of nucleic acids. In particular, the present invention relates to compositions and methods for generating and using hybridization probes.

BACKGROUND

FISH (fluorescence in situ hybridization) is a cytogenetic technique that is used to detect and localize the presence or absence of specific DNA sequences on chromosomes. FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence complementarity. Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosomes. FISH is often used for finding specific features in DNA for use in genetic counseling, medicine, and species identification. FISH can also be used to detect and localize specific RNA targets (mRNA, lncRNA and miRNA) in cells, circulating tumor cells, and tissue samples. In this context, it can help define the spatial-temporal patterns of gene expression within cells and tissues.

Human genomic DNA is a mixture of unique sequences and repetitive sequences that are present in multiple copies throughout the genome. In some applications, it is desirable to generate hybridization probes that anneal only to unique sequences of interest on a chromosome. Preparation of unique sequence probes is confounded by the presence of numerous classes of repetitive sequences throughout the genome of the organism (Hood et al., Molecular Biology of Eucaryotic Cells (Benjamin/Cummings Publishing Company, Menlo Park, Calif. 1975). The presence of repetitive sequences in hybridization probes reduces the specificity of the probes because portions of the probe bind to other repetitive sequences found outside the sequence of interest. Thus, to ensure binding of hybridization probes to a specific sequence of interest, probes lacking repetitive sequences are needed.

Recent contributions have addressed this question by inhibiting hybridization of the repetitive sequences with the use of unlabeled blocking nucleic acids (U.S. Pat. No. 5,447,841 and U.S. Pat. No. 6,596,479). Use of blocking nucleic acids in hybridizations is expensive, does not completely prevent hybridization of the repetitive sequences, and can distort genomic hybridization patterns (Newkirk et al., “Distortion of quantitative genomic and expression hybridization by Cot-1 DNA: mitigation of this effect,” Nucleic Acids Res. vol 33 (22):el91 (2005)). Thus, methods that prevent hybridization of repeat sequences without the use of blocking DNA are desirable for optimal hybridization.

One means to achieve this is to remove unwanted repeat segments from the hybridization probes prior to hybridization. Techniques involving the removal of highly repetitive sequences have been previously described. Absorbents, like hydroxyapatite, provide a means to remove highly repetitive sequences from extracted DNA. Hyroxyapatite chromatography fractionates DNA on the basis of duplex re-association conditions, such as temperature, salt concentration, or other stringencies. This procedure is cumbersome and varies with different sequences. Repeat DNA can also be removed by hybridization to immobilized DNA (Brison et al., “General Methods for Cloning Amplified DNA by Differential Screening with Genomic Probes,” Molecular and Cellular Biology, Vol. 2, pp. 578-587 (1982)). In all of these procedures, the physical removal of the repetitive sequences will depend upon the strict optimization of conditions with inherent variations based upon the base composition of the DNA sequence.

Several other methods to remove repetitive sequences from hybridization probes have been described. One method involves using a cross-linking agent to cross-link repetitive sequences either to directly prevent hybridization of repetitive sequences or to prevent amplification of repeat sequences in a PCR reaction. (U.S. Pat. No. 6,406,850). Another method uses PCR assisted affinity chromatography to remove repeats from hybridization probes (U.S. Pat. No. 6,569,621). Both of these methods rely on the use of labeled DNA to remove repeat sequences which makes these processes complex and difficult to reproduce. Further, both methods are time consuming, requiring multiple rounds of repeat removal to produce functional probes suitable for use in fluorescent in situ hybridization (FISH) or other hybridization reactions requiring high target specificity.

Thus, methods for removing repetitive sequences from probes are desired.

SUMMARY OF THE INVENTION

Embodiments of the present technology provide compositions, kits, and systems for generating and using probes selectively generated or synthesized to exclude sequences of disinterest and/or include sequences of interest (e.g., substantially repeat-free nucleic acid probes). For example, in some embodiments, the present invention provides a method of generating a probe to a nucleic acid of interest, comprising: a) identifying regions of the nucleic acid target of interest substantially free of undesired sequences (e.g., free of repeats, non-conserved sequences, conserved sequences, GC rich sequences, AT rich sequences, secondary structure, or coding sequences) of the nucleic acid of interest that are at least 100 by in length (e.g., at least 100, 200, at least 300, or at least 400) and optionally no more than 20% different in length from each other (e.g., 20% or less, 10% or less, 5% or less, 4% or less, 3% or less, 3% or less, 1% or less, or identical lengths); and b) generating (e.g., via amplification, cloning, synthesis, or a combination thereof) a plurality of probe-containing nucleic acids corresponding to the regions substantially free of undesired sequence. In some embodiments, the method further comprises one or more of the steps of c) fragmenting the probe-containing nucleic acids to generate probes; and d) further amplifying a subset of the probes to generate probes substantially free of undesired sequences (e.g., ISH probes lacking, for example, undesired repeat sequences). In some embodiments, the method further comprises the step of d) separating probes based on size. In some embodiments, the separating is conducted using chromatography or electrophoresis. In some embodiments, the method further comprises the step of isolating a subset of the probes. In some embodiments, the subset is based on size of the separated nucleic acid. In some embodiments, the probes are attached to nucleic acid adaptors. In some embodiments, the adaptors are amplification primers. In some embodiments, the amplification primers are functionalized for downstream applications (e.g., by the addition of labels, binding sites, or restriction sites). In some embodiments, the probes are separated and a subset of the probes is isolated. In some embodiments, the amplification is PCR. In some embodiments, regions substantially free of undesired sequence are identified using computer software and a computer processor. In some embodiments, the of probe-containing nucleic acids are fragmented by sonication (although any of a variety of other chemical, physical, or other approaches may be used). In some embodiments, the separating is by electrophoresis or chromatography. In some embodiments, the fragments are from about 100 to 500 by in length, although other lengths may be used. In some embodiments, the probes are labeled (e.g., with a fluorescent label). In some embodiments, probes are 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% free of undesired nucleic acid sequences.

In some embodiments, the present invention provides a method of generating a probe to a nucleic acid of interest, comprising: a) identifying regions of the nucleic acid target of interest substantially free of undesired sequences that are at least 100 by in length; b) generating a plurality of probe-containing nucleic acids corresponding to the regions substantially free of undesired sequence; c) fragmenting the probe-containing nucleic acids to generate probes; d) attaching adaptors to the probes; and optionally e) further amplifying a subset of the probes.

Further embodiments provide a method of generating a probe to a nucleic acid of interest, comprising: a) identifying regions of the nucleic acid target of interest substantially free of undesired sequences that are at least 100 by in length; b) generating a plurality of probe-containing nucleic acids corresponding to the regions substantially free of undesired sequence; c) fragmenting the probe-containing nucleic acids to generate probes; and optionally d) further amplifying a subset of the probes.

Additional embodiments provide a method of generating a probe to a nucleic acid of interest, comprising: a) identifying regions of the nucleic acid target of interest substantially free of undesired sequences, wherein the undesired region is, for example, repeat sequence, non-conserved sequences, conserved sequences, GC rich sequences, AT rich sequences, secondary structure, or coding sequences that are at least 100 by in length; b) generating a plurality of probe-containing nucleic acids corresponding to the regions substantially free of undesired sequence; and c) fragmenting the probe-containing nucleic acids to generate probes; and optionally d) further amplifying a subset of the probes.

Yet other embodiments provide a method of generating a probe to a nucleic acid of interest, comprising: a) identifying regions of the nucleic acid target of interest substantially free of undesired sequences that are at least 100 by in length; b) generating a plurality of probe-containing nucleic acids corresponding to the regions substantially free of undesired sequence; c) fragmenting the probe-containing nucleic acids to generate probes; d) separating the probes by size; e) isolating a subset of the probes; and optionally f) further amplifying a subset of the probes.

Still other embodiments provide a method of generating a probe to a nucleic acid of interest, comprising: a) identifying regions of the nucleic acid target of interest substantially free of undesired sequences that are at least 100 by in length; b) generating a plurality of probe-containing nucleic acids corresponding to the regions substantially free of undesired sequence; c) fragmenting the probe-containing nucleic acids to generate probes; d) separating the probes by size; e) isolating a subset of the probes, wherein the subset comprises nucleic acids of 80 to 300 by in length (e.g., approximately 150 by in length); and optionally f) further amplifying a subset of the probes.

Additional embodiments provide a method of generating a probe to a nucleic acid of interest, comprising: a) identifying regions of the nucleic acid target of interest substantially free of undesired sequences that are at least 100 by in length; b) generating a plurality of probe-containing nucleic acids corresponding to the regions substantially free of undesired sequence; and c) fragmenting the probe-containing nucleic acids to generate probes; optionally d) further amplifying a subset of the probes generate a probe set; and e) performing a hybridization assay (e.g., ISH assay) with the probe set.

Further provided herein are a set of nucleic acid probes (e.g., hybridization probes (e.g., in situ hybridization (ISH) probes)) free of undesired sequences generated by the aforementioned methods and kits and systems comprising the probes. The present disclosure is not limited to a particular assay or target. In some embodiments, probes detect expression of an oncogene or chromosomal aneuploidy.

Additionally provided herein are methods of performing a hybridization assay, comprising contacting a target nucleic acid with a probe (e.g., an ISH probe) generated by the aforementioned method.

Also provided herein is the use of any a probe (e.g., a ISH probe) generated by the aforementioned method in a hybridization (e.g., ISH) assay.

Additional embodiments are described herein.

DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic of exemplary labeled probes of embodiments of the present invention.

FIG. 2 shows a map of the HER2 locus.

FIG. 3 shows amplification of repeat-free probes from the HER locus.

FIG. 4 shows fragmented probes from the HER2 locus.

FIG. 5 shows amplification of adapted HER2 probes.

FIG. 6 shows HER2 repeat-free probes.

FIG. 7 shows FISH hybridization of the HER2 locus using probes of embodiments of the present disclosure.

FIGS. 8A-8G show 80 by portions of the HER-2 gene selected for use in designing HER-2 probes.

FIG. 9 shows a map of an exemplary repeat-free p16 probe.

DEFINITIONS

As used herein, the term “substantially free of undesired nucleic acids” refers to a nucleic acid that is substantially free (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% free) of undesired nucleic acids. Undesired nucleic acids include, but are not limited to, repeated nucleic acids, non-conserved sequences, conserved sequences, GC rich sequences, AT rich sequences, secondary structure, or coding sequences

As used, the terms “substantially repeat free nucleic acid sequence” or “nucleic acids free of repeats” refer to a region of nucleic acid that is substantially free (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% free) of repeated nucleic acid sequence.

As used herein, the term “sample” is used in its broadest sense. In one sense, it is meant to include cells (e.g., human, bacterial, yeast, and fungi), an organism, a specimen or culture obtained from any source, as well as biological samples. Biological samples may be obtained from animals (including humans) and refers to a biological material or compositions found therein, including, but not limited to, bone marrow, blood, serum, platelet, plasma, interstitial fluid, urine, cerebrospinal fluid, nucleic acid, DNA, tissue, and purified or filtered forms thereof. Such examples are not however to be construed as limiting the sample types applicable to the present invention.

The term “label” as used herein refers to any atom or molecule that can be used to provide a detectable (preferably quantifiable) effect, and that can be attached to a nucleic acid or protein. Labels include but are not limited to dyes; radiolabels such as ³²P; binding moieties such as biotin; haptens such as digoxgenin; luminogenic, phosphorescent or fluorogenic moieties; and fluorescent dyes alone or in combination with moieties that can suppress or shift emission spectra by fluorescence resonance energy transfer (FRET). Labels may provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like. A label may be a charged moiety (positive or negative charge) or alternatively, may be charge neutral. Labels can include or consist of nucleic acid or protein sequence, so long as the sequence comprising the label is detectable. In some embodiments, nucleic acids are detected directly without a label (e.g., directly reading a sequence).

As used herein, the term “nucleic acid molecule” refers to any nucleic acid containing molecule, including but not limited to, DNA or RNA. The term encompasses sequences that include any of the known base analogs of DNA and RNA including, but not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methyl cytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methyl aminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

As used herein, the term “hybridization” is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, the T_mof the formed hybrid, and the G:C ratio within the nucleic acids. A single molecule that contains pairing of complementary nucleic acids within its structure is to be “self-hybridized.”

The term “primer” refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (e.g., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer should be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method. For example, in some embodiments, primers range from 10 to 100 or more nucleotides (e.g., 10-300, 15-250, 15-200, 15-150, 15-100, 15-90, 20-80, 20-70, 20-60, 20-50 nucleotides, etc.).

In some embodiments, primers comprise additional sequences that do not hybridize to the nucleic acid of interest. The term “primer” includes chemically modified primers, fluorescence-modified primers, functional primers (fusion primers), sequence specific primers, random primers, primers that have both specific and random sequences, and DNA and RNA primers

The term “amplifying” or “amplification” in the context of nucleic acids refers to the production of multiple copies of a polynucleotide, or a portion of the polynucleotide, typically starting from a small amount of the polynucleotide (e.g., as few as a single polynucleotide molecule), where the amplification products or amplicons are generally detectable. Amplification of polynucleotides encompasses a variety of chemical and enzymatic processes. The generation of multiple DNA copies from one or a few copies of a target or template DNA molecule during a polymerase chain reaction (PCR), rolling circle amplification (RCA), or a ligase chain reaction (LCR) are forms of amplification. Amplification is not limited to the strict duplication of the starting molecule. For example, the generation of multiple cDNA molecules from a limited amount of RNA in a sample using reverse transcription (RT)-PCR is a form of amplification. Furthermore, the generation of multiple RNA molecules from a single DNA molecule during the process of transcription is also a form of amplification.

As used herein, the term “solid support” is used in reference to any solid or stationary material to which reagents such as antibodies, antigens, and other test components are attached. Examples of solid supports include microscope slides, wells of microtiter plates, coverslips, beads, particles, cell culture flasks, as well as many other suitable items.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Embodiments of the present technology provide compositions and methods for generating probes substantially free of undesired sequences (e.g., FISH probes lacking undesired sequences, such as repeat sequences) that solve the limitations of existing methods of removing undesired sequences from probes by providing multistep or otherwise burdensome methods.

Embodiments of the present invention are illustrated for use in generating repeat free ISH probes. One of skill in art understands that the disclosed methods can also be applied to other undesired sequences and other probe applications.

The presence of repeat sequence in FISH probes leads to background signals in other loci containing similar repeats. Additionally it increases the bulk of the FISH probe unnecessarily, leading to wasted material. Finally, regardless of the repeat sequence, FISH probes are typically prepared from DNA generated directly from BAC sequences over 100 kb in length. The methods provided herein allow selection of non-repeat sequence, specific to the genomic region of interest, circumventing potential issues with availability of BACs covering the genomic region of interest and overcomes disadvantages of existing methods of generating repeat-free probes (See e.g., Rogan et al., Genome Research 11:1086-1094, 2001; Commercially available probes from Kreatech, Durham, N.C.; Sealey et al. Nuc. Acid. Res. Volume 13 Number 6 1985; Dorman et al., Nucleic Acids Research, 2013, Vol. 41, No. 7; Boyle et al., Chromosome Res. 2011 October; 19(7):901-9; and Craig et al., Hum Genet (1997) 100: 472-476).

The probes described herein provide the following advantages over existing probes: decrease in interference from repeat sequence; eliminates the need for human DNA blocker; faster hybridization times; higher hybridization temperature (e.g., easier probe removal and more uniform hybridization temperatures), resulting in faster hybridization times; preparation via amplification (e.g., PCR) gives faster, less expensive, more reliable manufacture; and, once made, the template finds use in scale up and manufacturing applications.

I. Generation of Probes

Exemplary methods of generating probes substantially free of undesired sequences are described below. Embodiments of the present invention are illustrated for use in generating ISH (e.g., FISH) probes. One of skill in art understands that the disclosed methods can also be applied to other undesired sequences and other probe applications.

Embodiments of the present technology provide compositions, kits, and systems for generating and using probes selectively generated or synthesized to exclude sequences of disinterest and/or include sequences of interest (e.g., substantially repeat-free nucleic acid probes). For example, in some embodiments, the present invention provides a method of generating a probe to a nucleic acid of interest, comprising: a) identifying regions of the nucleic acid target of interest substantially free of undesired sequences (e.g., free of repeats, non-conserved sequences, conserved sequences, GC rich sequences, AT rich sequences, secondary structure, or coding sequences) of the nucleic acid of interest that are at least 100 by in length (e.g., at least 100, 200, at least 300, or at least 400) and optionally no more than 20% different in length from each other (e.g., 20% or less, 10% or less, 5% or less, 4% or less, 3% or less, 3% or less, 1% or less, or identical lengths); and b) generating (e.g., via amplification, cloning, synthesis, or a combination thereof) a plurality of probe-containing nucleic acids corresponding to the regions substantially free of undesired sequence. In some embodiments, the method further comprises one or more of the steps of c) fragmenting the probe-containing nucleic acids to generate probes; and d) further amplifying a subset of the probes to generate probes substantially free of undesired sequences (e.g., ISH probes lacking, for example, undesired repeat sequences). In some embodiments, the method further comprises the step of d) separating probes based on size. In some embodiments, the separating is conducted using chromatography or electrophoresis. In some embodiments, the method further comprises the step of isolating a subset of the probes. In some embodiments, the subset is based on size of the separated nucleic acid. In some embodiments, the probes are attached to nucleic acid adaptors. In some embodiments, the adaptors are amplification primers. In some embodiments, the amplification primers are functionalized for downstream applications (e.g., by the addition of labels, binding sites, or restriction sites). In some embodiments, the probes are separated and a subset of the probes is isolated. In some embodiments, the amplification is PCR. In some embodiments, regions substantially free of undesired sequence are identified using computer software and a computer processor. In some embodiments, the probe-containing nucleic acids are fragmented by sonication (although any of a variety of other chemical, physical, or other approaches may be used). In some embodiments, the separating is by electrophoresis or chromatography. In some embodiments, the fragments are from about 100 to 500 by in length, although other lengths may be used. In some embodiments, the probes are labeled (e.g., with a fluorescent label). In some embodiments, probes are 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% free of undesired nucleic acid sequences.

In some embodiments, the probes are approximately 50 to 1000 by in length. For example, in some embodiments, probes are 50 to 900 bp, 50 to 800 bp, 50 to 700 bp, 50 to 600 bp, 50 to 500 bp, 50 to 450 bp, 50 to 400 bp, 50 to 350 bp, 50 to 300 bp, 50 to 250 bp, 50 to 200 bp, 50 to 150 bp, 50 to 100 bp, 80 to 900 bp, 80 to 800 bp, 80 to 700 bp, 80 to 600 bp, 80 to 500 bp, 80 to 450 bp, 80 to 400 bp, 80 to 350 bp, 80 to 300 bp, 80 to 250 bp, 80 to 200 bp, 80 to 150 bp, 80 to 100 bp, 100 to 900 bp, 100 to 800 bp, 100 to 700 bp, 100 to 600 bp, 100 to 500 bp, 100 to 450 bp, 100 to 400 bp, 100 to 350 bp, 100 to 300 bp, 100 to 250 bp, 100 to 200 bp, 100 to 150 bp, 150 to 900 bp, 150 to 800 bp, 150 to 700 bp, 150 to 600 bp, 150 to 500 bp, 150 to 450 bp, 150 to 400 bp, 150 to 350 bp, 150 to 300 bp, 150 to 250 bp, 150 to 200 bp, 150 to 150 bp, or 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150 bp. In some embodiments, probes are 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% free of undesired nucleic acid sequences.

Additional embodiments provide a method of generating a probe to a nucleic acid of interest, comprising: a) identifying regions of the nucleic acid target of interest substantially free of undesired sequences that are at least 100 by in length; b) generating a plurality of probe-containing nucleic acids corresponding to the regions substantially free of undesired sequence; and c) fragmenting the probe-containing nucleic acids to generate probes; optionally d) further amplifying a subset of the probes generate a probe set; and e) performing a hybridization assay (e.g., ISH assay such as FISH) with the probe set.

Further provided herein are a set of nucleic acid probes (e.g., ISH probes) free of undesired sequences generated by the aforementioned methods and kits and systems comprising the probes.

Additionally provided herein are methods of performing a hybridization assay, comprising contacting a target nucleic acid with a probe (e.g., a ISH probe) generated by the aforementioned method.

Also provided herein is the use of any a probe (e.g., a ISH probe) generated by the aforementioned method in a hybridization (e.g., ISH) assay.

Exemplary methods of generating probes are described in detail below.

A. Probes

The present invention provides, in some embodiments, methods of generating probe libraries. In some embodiments, probes are between approximately 100 and 400 by in length (e.g., between 100 and 300 by in length).

In some embodiments, libraries of probes are generated that are complementary to different regions of a target sequence. In some embodiments, all of the probes in a library are of similar length (e.g., within 1%, 2%, 3%, 4%, or 5%, 10%, 20% or identical in length).

Probes can comprise any number of modified bases, modified backbones, inclusion of minor groove binders, and labels (e.g., as described in more detail below). Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2′-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).

B. Identification of undesired sequences

The present invention is not limited to a particular type of undesired sequences. In some embodiments, undesired sequences are, for example, repeat sequence, non-conserved sequences, conserved sequences, GC rich sequences, AT rich sequences, secondary structure, and coding sequences. In some preferred embodiments, repeat-free sequences are removed in order to optimize probe binding.

In some embodiments, substantially repeat-free or other undesired segments of genomic DNA in a region of interest are first identified. In some embodiments, contiguous repeat-free segments of genomic DNA in a region of interest are identified by bioinformatics methods. The present invention is not limited to a particular bioinformatics method. In some embodiments, commercial software packages such as, for example the RepeatMasker function of UCSC Genome Browser (available from The National Cancer Institute's Center for Biomedical Informatics and Information Technology) are utilized, although other commercial or non-commercial software packages are specifically contemplated.

In some embodiments, the Repeat-Masker function of Genome Browser is used to distinguish repeat from nonrepeat portions by presenting repeat sequence in lower case and nonrepeat in upper case, and the sequence downloaded. External software programs or manual examination of sequence information are used to remove the repeat portion of the sequence and to present the stretches of contiguous nonrepeat sequence in order of their length. The shorter stretches (usually less than 300 bp) are discarded, and a number of the longer stretches are identified such that their combined sequence length is sufficient to prepare a probe with acceptable labeling intensity for the intended use.

C. Generation of Probes

Following identification of undesired sequences, probes are designed that are substantially free of undesired sequences. Probes may be generated using any suitable method. In some embodiments, probes are amplified using the method described below.

In some embodiments, libraries of probes are synthesized. In some embodiments, synthetic probes comprise a region of complementarity to the target sequence and a label region that is not-complementary to the target sequence (See e.g., FIG. 1). In some embodiments, the labeled regions are identical across the probe library. In some embodiments, probes are generated with a labeled region that is branched or another non-linear configuration.

In some embodiments, oligonucleotides for use in amplification methods or probe generation are synthesized. Exemplary methods for oligonucleotide synthesis are described herein. To obtain the desired oligonucleotide, the building blocks are sequentially coupled to the growing oligonucleotide chain in the order required by the sequence of the product. The process has been fully automated since the late 1970s. Upon the completion of the chain assembly, the product is released from the solid phase to solution, deprotected, and collected. Products are often isolated by high-performance liquid chromatography (HPLC) to obtain the desired oligonucleotides in high purity. Typically, synthetic oligonucleotides are single-stranded DNA or RNA molecules around 15-25 bases in length.

In some embodiments, the selectivity and the rate of the formation of internucleosidic linkages is improved by using 3′-O—(N,N-diisopropyl phosphoramidite) derivatives of nucleosides (nucleoside phosphoramidites) that serve as building blocks in phosphite triester methodology. To prevent undesired side reactions, all other functional groups present in nucleosides are rendered unreactive (protected) by attaching protecting groups. Upon the completion of the oligonucleotide chain assembly, all the protecting groups are removed to yield the desired oligonucleotides.

Exemplary protecting groups and nucleoside phosphoramidite building blocks include, but are not limited to, acid-labile DMT (4,4′-dimethoxytrityl) protecting groups. Thymine and uracil, nucleic bases of thymidine and uridine, respectively, do not have exocyclic amino groups and hence do not require any protection.

Although the nucleic base of guanosine and 2′-deoxyguanosine does have an exocyclic amino group, its basicity is low to an extent that it does not react with phosphoramidites under the conditions of the coupling reaction. However, a phosphoramidite derived from the N2-unprotected 5′-O-DMT-2′-deoxyguanosine is poorly soluble in acetonitrile, the solvent commonly used in oligonucleotide synthesis. In contrast, the N2-protected versions of the same compound dissolve in acetonitrile well and hence are widely used. Nucleic bases adenine and cytosine bear the exocyclic amino groups reactive with the activated phosphoramidites under the conditions of the coupling reaction. By the use of additional steps in the synthetic cycle or alternative coupling agents and solvent systems, the oligonucleotide chain assembly are carried out using dA and dC phosphoramidites with unprotected amino groups. In some embodiments, exocyclic amino groups in nucleosides are kept permanently protected over the entire length of the oligonucleotide chain assembly.

The protection of the exocyclic amino groups is generally orthogonal to that of the 5′-hydroxy group because the latter is removed at the end of each synthetic cycle. The simplest to implement and hence the most widely accepted is the strategy where the exocyclic amino groups bear a base-labile protection. Most often, two protection schemes are used.

In some embodiments, Bz (benzoyl) protection is used for A, dA, C, and dC, while G and dG are protected with isobutyryl group. More recently, Ac (acetyl) group is often used to protect C and dC.

In the second, mild protection scheme, A and dA are protected with isobutyryl or phenoxyacetyl groups (PAC). C and dC bear acetyl protection, and G and dG are protected with 4-isopropylphenoxyacetyl (iPr-PAC) or dimethylformamidino (dmf) groups. Mild protecting groups are removed more readily than the standard protecting groups. However, the phosphoramidites bearing these groups are less stable when stored in solution.

In some embodiments, the phosphite group is protected by a base-labile 2-cyanoethyl group. Once a phosphoramidite has been coupled to the solid support-bound oligonucleotide and the phosphite moieties have been converted to the P(V) species, the presence of the phosphate protection is not mandatory for the successful conducting of further coupling reactions.

Non-nucleoside phosphoramidites are the phosphoramidite reagents designed to introduce various functionalities at the termini of synthetic oligonucleotides or between nucleotide residues in the middle of the sequence. In order to be introduced inside the sequence, a non-nucleosidic modifier has to possess at least two hydroxy groups, one of which is often protected with the DMT group while the other bears the reactive phosphoramidite moiety.

Non-nucleosidic phosphoramidites are used to introduce desired groups that are not available in natural nucleosides or that can be introduced more readily using simpler chemical designs.

Oligonucleotide synthesis is carried out by a stepwise addition of nucleotide residues to the 5′-terminus of the growing chain until the desired sequence is assembled. Each addition is referred to as a synthetic cycle and consists of four chemical reactions:

Step 1: De-Blocking (Detritylation)

The DMT group is removed with a solution of an acid, such as 2% trichloroacetic acid (TCA) or 3% dichloroacetic acid (DCA), in an inert solvent (dichloromethane or toluene). The orange-colored DMT cation formed is washed out; the step results in the solid support-bound oligonucleotide precursor bearing a free 5′-terminal hydroxyl group. Conducting detritylation for an extended time or with stronger than recommended solutions of acids leads to depurination of solid support-bound oligonucleotide and thus reduces the yield of the desired full-length product.

A solution of nucleoside phosphoramidite (or a mixture of several phosphoramidites) in acetonitrile is next activated by an acidic azole catalyst, 1H-tetrazole, 2-ethylthiotetrazole, 2-benzylthiotetrazole, 4,5-dicyanoimidazole, or a number of similar compounds. The mixing is usually very brief and occurs in fluid lines of oligonucleotide synthesizers while the components are being delivered to the reactors containing solid support. The activated phosphoramidite in 1.5-20-fold excess over the support-bound material is then brought in contact with the starting solid support (first coupling) or a support-bound oligonucleotide precursor (following couplings) whose 5′-hydroxy group reacts with the activated phosphoramidite moiety of the incoming nucleoside phosphoramidite to form a phosphite triester linkage. The coupling of 2′-deoxynucleoside phosphoramidites is very rapid and requires, on small scale, about 20 s for its completion. In contrast, sterically hindered 2′-O-protected ribonucleoside phosphoramidites utilize longer times to be coupled in high yields. The reaction is also highly sensitive to the presence of water, particularly when dilute solutions of phosphoramidites are used, and is commonly carried out in anhydrous acetonitrile. Generally, the larger the scale of the synthesis, the lower the excess and the higher the concentration of the phosphoramidites is used. In contrast, the concentration of the activator is primarily determined by its solubility in acetonitrile and is irrespective of the scale of the synthesis. Upon the completion of the coupling, any unbound reagents and by-products are removed by washing.

The capping step is performed by treating the solid support-bound material with a mixture of acetic anhydride and 1-methylimidazole or, less often, DMAP as catalysts and, in the phosphoramidite method, serves two purposes.

After the completion of the coupling reaction, a small percentage of the solid support-bound 5′-OH groups (0.1 to 1%) remains unreacted and needs to be permanently blocked from further chain elongation to prevent the formation of oligonucleotides with an internal base deletion commonly referred to as (n−1) shortmers. The unreacted 5′-hydroxy groups are, to a large extent, acetylated by the capping mixture.

It has also been reported that phosphoramidites activated with 1H-tetrazole react, to a small extent, with the O6 position of guanosine. Upon oxidation with I2/water, this side product, possibly via O6-N7 migration, undergoes depurination. The apurinic sites thus formed are readily cleaved in the course of the final deprotection of the oligonucleotide under the basic conditions (see below) to give two shorter oligonucleotides thus reducing the yield of the full-length product. The O6 modifications are rapidly removed by treatment with the capping reagent as long as the capping step is performed prior to oxidation with I2/water.

The synthesis of oligonucleotide phosphorothioates (OPS) does not involve the oxidation with I2/water, and, respectively, does not suffer from the side reaction described above. On the other hand, if the capping step is performed prior to sulfurization, the solid support may contain the residual acetic anhydride and N-methylimidazole left after the capping step. The capping mixture interferes with the sulfur transfer reaction, which results in the extensive formation of the phosphate triester internucleosidic linkages in place of the desired PS triesters. Therefore, for the synthesis of OPS, it is advisable to conduct the sulfurization step prior to the capping step.

The newly formed tricoordinated phosphite triester linkage is not natural and is of limited stability under the conditions of oligonucleotide synthesis. The treatment of the support-bound material with iodine and water in the presence of a weak base (pyridine, lutidine, or collidine) oxidizes the phosphite triester into a tetracoordinated phosphate triester, a protected precursor of the naturally occurring phosphate diester internucleosidic linkage. Oxidation may be carried out under anhydrous conditions using tert-Butyl hydroperoxide or, more efficiently, (1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO). The step of oxidation is substituted with a sulfurization step to obtain oligonucleotide phosphorothioates. In the latter case, the sulfurization step is best carried out prior to capping.

In solid-phase synthesis, an oligonucleotide being assembled is covalently bound, via its 3′-terminal hydroxy group, to a solid support material and remains attached to it over the entire course of the chain assembly. The solid support is contained in columns whose dimensions depend on the scale of synthesis and may vary between 0.05 mL and several liters. The overwhelming majority of oligonucleotides are synthesized on small scale ranging from 40 nmol to 1 μmol. More recently, high-throughput oligonucleotide synthesis where the solid support is contained in the wells of multi-well plates (most often, 96 or 384 wells per plate) became a method of choice for parallel synthesis of oligonucleotides on small scale. At the end of the chain assembly, the oligonucleotide is released from the solid support and is eluted from the column or the well.

In contrast to organic solid-phase synthesis and peptide synthesis, the synthesis of oligonucleotides proceeds best on non-swellable or low-swellable solid supports. The two most often used solid-phase materials are controlled pore glass (CPG) and macroporous polystyrene (MPPS).

CPG is commonly defined by its pore size. In oligonucleotide chemistry, pore sizes of 500, 1000, 1500, 2000, and 3000 Å are used to allow the preparation of about 50, 80, 100, 150, and 200-mer oligonucleotides, respectively. To make native CPG suitable for further processing, the surface of the material is treated with (3-aminopropyl)triethoxysilane to give aminopropyl CPG. The aminopropyl arm may be further extended to result in long chain aminoalkyl (LCAA) CPG. The amino group is then used as an anchoring point for linkers suitable for oligonucleotide synthesis (see below).

MPPS suitable for oligonucleotide synthesis is a low-swellable, highly cross-linked polystyrene obtained by polymerization of divinylbenzene (min 60%), styrene, and 4-chloromethylstyrene in the presence of a porogeneous agent. The macroporous chloromethyl MPPS obtained is converted to aminomethyl MPPS.

To make the solid support material suitable for oligonucleotide synthesis, non-nucleosidic linkers or nucleoside succinates are covalently attached to the reactive amino groups in aminopropyl CPG, LCAA CPG, or aminomethyl MPPS. The remaining unreacted amino groups are capped with acetic anhydride. Typically, three conceptually different groups of solid supports are used.

In a more recent, more convenient, and more widely used method, the synthesis starts with the universal support where a non-nucleosidic linker is attached to the solid support material. A phosphoramidite respective to the 3′-terminal nucleoside residue is coupled to the universal solid support in the first synthetic cycle of oligonucleotide chain assembly using the standard protocols. The chain assembly is then continued until the completion, after which the solid support-bound oligonucleotide is deprotected. The characteristic feature of the universal solid supports is that the release of the oligonucleotides occurs by the hydrolytic cleavage of a P—O bond that attaches the 3′-O of the 3′-terminal nucleotide residue to the universal linker as shown in Scheme 6. The critical advantage of this approach is that the same solid support is used irrespectively of the sequence of the oligonucleotide to be synthesized. For the complete removal of the linker and the 3′-terminal phosphate from the assembled oligonucleotide, the solid support 1 and several similar solid supports require gaseous ammonia, aqueous ammonium hydroxide, aqueous methylamine, or their mixture and are commercially available. The solid support utilizes a solution of ammonia in anhydrous methanol and is also commercially available.

In general, the 3′-hydroxy group of the 3′-terminal nucleoside residue is attached to the solid support via, most often, 3′-O-succinyl arm as in compound 3. The oligonucleotide chain assembly starts with the coupling of a phosphoramidite building block respective to the nucleotide residue second from the 3′-terminus. The 3′-terminal hydroxy group in oligonucleotides synthesized on nucleosidic solid supports is deprotected under the conditions somewhat milder than those applicable for universal solid supports. However, the fact that a nucleosidic solid support has to be selected in a sequence-specific manner reduces the throughput of the entire synthetic process and increases the likelihood of human error.

Oligonucleotide phosphorothioates (OPS) are modified oligonucleotides where one of the oxygen atoms in the phosphate moiety is replaced by sulfur. Only the phosphorothioates having sulfur at a non-bridging position are widely used and are available commercially. The replacement of the non-bridging oxygen with sulfur creates a new center of chirality at phosphorus. In a simple case of a dinucleotide, this results in the formation of a diastereomeric pair of Sp- and Rp-dinucleoside monophosphorothioates. In a n-mer oligonucleotide where all (n−1) internucleosidic linkages are phosphorothioate linkages, the number of diastereomers m is calculated as m=2(n−1). Being non-natural analogs of nucleic acids, OPS are substantially more stable towards hydrolysis by nucleases, the class of enzymes that destroy nucleic acids by breaking the bridging P—O bond of the phosphodiester moiety. This property determines the use of OPS as antisense oligonucleotides in in vitro and in vivo applications where the extensive exposure to nucleases is inevitable. Similarly, to improve the stability of siRNA, at least one phosphorothioate linkage is often introduced at the 3′-terminus of both sense and antisense strands. In chirally pure OPS, all-Sp diastereomers are more stable to enzymatic degradation than their all-Rp analogs. However, the preparation of chirally pure OPS remains a synthetic challenge. In laboratory practice, mixtures of diastereomers of OPS are commonly used.

Synthesis of OPS is very similar to that of natural oligonucleotides. The difference is that the oxidation step is replaced by sulfur transfer reaction (sulfurization) and that the capping step is performed after the sulfurization. Of many reported reagents capable of the efficient sulfur transfer, only three are commercially available:

3-(Dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-3-thione, DDTT (3) provides rapid kinetics of sulfurization and high stability in solution. 3H-1,2-benzodithiol-3-one 1,1-dioxide (4) also known as Beaucage reagent displays a better solubility in acetonitrile and short reaction times. However, the reagent is of limited stability in solution and is less efficient in sulfurizing RNA linkages.

N,N,N′N′-Tetraethylthiuram disulfide (TETD) is soluble in acetonitrile and is commercially available. However, the sulfurization reaction of an internucleosidic DNA linkage with TETD requires 15 min.

In the past, oligonucleotide synthesis was carried out manually in solution or on solid phase. The solid phase synthesis was implemented using, as containers for the solid phase, miniature glass columns similar in their shape to low-pressure chromatography columns or syringes equipped with porous filters. Currently, solid-phase oligonucleotide synthesis is carried out automatically using computer-controlled instruments (oligonucleotide synthesizers) and is technically implemented in column, multi-well plate, and array formats. The column format is best suited for research and large scale applications where a high-throughput is not required. Multi-well plate format is designed specifically for high-throughput synthesis on small scale to satisfy the growing demand of industry and academia for synthetic oligonucleotides. A number of oligonucleotide synthesizers for small scale synthesis and medium to large scale synthesis are available commercially.

Amplification methods are described below, although other methods may be used. Next, amplification (e.g., PCR) primers are designed to amplify stretches of sequence from the longest of the repeat-free segments identified using bioinformatics methods. In some embodiments, such segments are amplified using genomic or genome derived BAC DNA as an amplification template. In some embodiments, (e.g., in the case of longer stretches) multiple overlapping primer sets are used.

Illustrative non-limiting examples of nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), transcription-mediated amplification (TMA), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence based amplification (NASBA).

In general, amplification methods utilize a DNA polymerase, a primer, and dNTPs. Exemplary DNA polymerases include, but are not limited to, phi29 DNA Polymerase, Taq DNA polymerase, DNA polymerase I, T7 DNA Polymerase, T7 DNA Polymerase, T4 DNA Polymerase, Pfu DNA Polymerase, and Bsm DNA Polymerase.

The polymerase chain reaction (U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159 and 4,965,188, each of which is herein incorporated by reference in its entirety), commonly referred to as PCR, uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase copy numbers of a target nucleic acid sequence. In a variation called RT-PCR, reverse transcriptase (RT) is used to make a complementary DNA (cDNA) from mRNA, and the cDNA is then amplified by PCR to produce multiple copies of DNA. For other various permutations of PCR see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159; Mullis et al., Meth. Enzymol. 155: 335 (1987); and, Murakawa et al., DNA 7: 287 (1988), each of which is herein incorporated by reference in its entirety.

Transcription mediated amplification (U.S. Pat. Nos. 5,480,784 and 5,399,491, each of which is herein incorporated by reference in its entirety), commonly referred to as TMA, synthesizes multiple copies of a target nucleic acid sequence autocatalytically under conditions of substantially constant temperature, ionic strength, and pH in which multiple RNA copies of the target sequence autocatalytically generate additional copies. See, e.g., U.S. Pat. Nos. 5,399,491 and 5,824,518, each of which is herein incorporated by reference in its entirety. In a variation described in U.S. Publ. No. 20060046265 (herein incorporated by reference in its entirety), TMA optionally incorporates the use of blocking moieties, terminating moieties, and other modifying moieties to improve TMA process sensitivity and accuracy.

The ligase chain reaction (Weiss, R., Science 254: 1292 (1991), herein incorporated by reference in its entirety), commonly referred to as LCR, uses two sets of complementary DNA oligonucleotides that hybridize to adjacent regions of the target nucleic acid. The DNA oligonucleotides are covalently linked by a DNA ligase in repeated cycles of thermal denaturation, hybridization and ligation to produce a detectable double-stranded ligated oligonucleotide product.

Strand displacement amplification (Walker, G. et al., Proc. Natl. Acad. Sci. USA 89: 392-396 (1992); U.S. Pat. Nos. 5,270,184 and 5,455,166, each of which is herein incorporated by reference in its entirety), commonly referred to as SDA, uses cycles of annealing pairs of primer sequences to opposite strands of a target sequence, primer extension in the presence of a dNTPs to produce a duplex hemiphosphorothioated primer extension product, endonuclease-mediated nicking of a hemimodified restriction endonuclease recognition site, and polymerase-mediated primer extension from the 3′ end of the nick to displace an existing strand and produce a strand for the next round of primer annealing, nicking and strand displacement, resulting in geometric amplification of product. Thermophilic SDA (tSDA) uses thermophilic endonucleases and polymerases at higher temperatures in essentially the same method (EP Pat. No. 0 684 315).

Other amplification methods include, for example: nucleic acid sequence based amplification (U.S. Pat. No. 5,130,238, herein incorporated by reference in its entirety), commonly referred to as NASBA; one that uses an RNA replicase to amplify the probe molecule itself (Lizardi et al., BioTechnol. 6: 1197 (1988), herein incorporated by reference in its entirety), commonly referred to as Qβ replicase; a transcription based amplification method (Kwoh et al., Proc. Natl. Acad. Sci. USA 86:1173 (1989)); and, self-sustained sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA 87: 1874 (1990), each of which is herein incorporated by reference in its entirety). For further discussion of known amplification methods see Persing, David H., “In Vitro Nucleic Acid Amplification Techniques” in Diagnostic Medical Microbiology: Principles and Applications (Persing et al., Eds.), pp. 51-87 (American Society for Microbiology, Washington, D.C. (1993)).

In some embodiments, amplification is isothermal amplification. In some embodiments, amplification methods are solid-phase amplification, polony amplification, colony amplification, emulsion PCR, bead RCA, surface RCA, surface SDA, etc., as will be recognized by one of skill in the art. In some embodiments, amplification methods that results in amplification of free DNA molecules in solution or tethered to a suitable matrix by only one end of the DNA molecule are used. In some embodiments, methods that rely on bridge PCR, where both PCR primers are attached to a surface (see, e.g., WO 2000/018957, U.S. Pat. Nos. 7,972,820; 7,790,418 and Adessi et al., Nucleic Acids Research (2000): 28(20): E87; each of which are herein incorporated by reference) are used. In some cases the methods of the invention can create a “polymerase colony technology”, or “polony”, referring to a multiplex amplification that maintains spatial clustering of identical amplicons (see Harvard Molecular Technology Group and Lipper Center for Computational Genetics website). These include, for example, in situ polonies (Mitra and Church, Nucleic Acid Research 27, e34, Dec. 15, 1999), in situ rolling circle amplification (RCA) (Lizardi et al., Nature Genetics 19, 225, July 1998), bridge PCR (U.S. Pat. No. 5,641,658), picotiter PCR (Leamon et al., Electrophoresis 24, 3769, November 2003), and emulsion PCR (Dressman et al., PNAS 100, 8817, Jul. 22, 2003).

Examples of nucleic acid polymerases suitable for use in embodiments of the present invention include, but are not limited to, DNA polymerase (Klenow fragment, T4 DNA polymerase), thermostable DNA polymerases (Perler F. B. et al., Adv. Protein Chem. 1996, 48:377-435) identified and cloned in a variety of thermostable bacteria (such as Taq, VENT, Pfu, Tfl DNA polymerases) as well as their genetically modified derivatives (TaqGold, VENTexo, Pfu exo). Preferably the nucleic acid polymerase used for colony primer extension is stable under temperature at which the primer and template hybridization results enough specific to avoid incomplete or spurious amplifications of the template.

The amplification solution contains preferably, deoxyribonucleoside triphosphates, for example dATP, dTTP, dCTP, dGTP, naturally or non-naturally occurring, for example modified with a fluorescent or radioactive group. A large variety of synthetically modified nucleic acids have been developed for chemical and biological methods in order to increase the detectability and/or the functional diversity of nucleic acids. These functionalized/modified molecules (e.g., nucleotide analogs) can be fully compatible with natural polymerizing enzymes, maintaining the base pairing and replication properties of the natural counterparts, as recently reviewed (Thum O et al., Angew. Chem. Int. Ed. 2001, 40 (21): 3990-3993).

Other components of the amplification solution are added consequently to the choice of the nucleic acid polymerase, and they are essentially corresponding to compounds known in the art as being effective to support the activity of each polymerase. The concentration of compounds like dimethyl sulfoxide (DMSO), Bovine Serum Albumin (BSA), poly-ethylene glycol (PEG), Betaine, Triton X-100, or MgCl₂is well known in the prior art as being important to have an optimal amplification, and therefore the operator can easily adjust such concentrations for the methods of the present invention on the basis of the examples presented hereafter.

D. Fragmentation

In some embodiments, before or after amplification, the DNA is then fragmented (e.g., by sonication or other suitable method such as DNAse I) to lengths ranging from approximately 50 to 5000 by (e.g., 50 to 4000, 50 to 3000, 50 to 2500, 50 to 2000, 50 to 1500, 50 to 1000, 100 to 5000, 100 to 4000, 1000 to 3000, 100 to 2500, 100 to 2000, 100 to 1500, 100 to 1000, or 100-500 bp), and the resultant repeat-free DNA library attached (e.g., via ligation, chemical, extension reaction, etc.) to adapters. In some preferred embodiments, probes are approximately 150 by (e.g., 50 to 900 bp, 50 to 800 bp, 50 to 700 bp, 50 to 600 bp, 50 to 500 bp, 50 to 450 bp, 50 to 400 bp, 50 to 350 bp, 50 to 300 bp, 50 to 250 bp, 50 to 200 bp, 50 to 150 bp, 50 to 100 bp, 80 to 900 bp, 80 to 800 bp, 80 to 700 bp, 80 to 600 bp, 80 to 500 bp, 80 to 450 bp, 80 to 400 bp, 80 to 350 bp, 80 to 300 bp, 80 to 250 bp, 80 to 200 bp, 80 to 150 bp, 80 to 100 bp, 100 to 900 bp, 100 to 800 bp, 100 to 700 bp, 100 to 600 bp, 100 to 500 bp, 100 to 450 bp, 100 to 400 bp, 100 to 350 bp, 100 to 300 bp, 100 to 250 bp, 100 to 200 bp, 100 to 150 bp, 150 to 900 bp, 150 to 800 bp, 150 to 700 bp, 150 to 600 bp, 150 to 500 bp, 150 to 450 bp, 150 to 400 bp, 150 to 350 bp, 150 to 300 bp, 150 to 250 bp, 150 to 200 bp, 150 to 150 bp, or 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150 bp, etc.). In some embodiments, the adapted library is then fractionated (e.g., by electrophoresis, chromatography, or other separation method) to give a pool containing all the selected sequence at a variety of fragment sizes. Fractions (e.g., gel slices) corresponding to the desired fragment sizes are isolated.

E. Amplification

In some embodiments, fractions of the desired size are used as templates with amplification (e.g., PCR or another method described herein) primers corresponding to the adapters under preparatory amplification conditions.

All the chosen repeat-free sequence are represented in the amplified library. The probes (e.g., isolated after fragmentation or optional further amplification) are then labeled (e.g., with a fluorescent label, biotin, quantum dot label, or labels for colorimetric or silver stain detection) for use (e.g., as a FISH reagent).

In some embodiments, libraries of probes are cloned into expression vectors (e.g., one or more probes per vector). In some embodiments, such expression vectors find use in the future generation of libraries (e.g., via amplification or expression).

II. Uses of Probes

The probes described herein find use in a variety of diagnostic, research, clinical, and screening applications. The described nucleic acid hybridization probes have broad utility for use in all forms of nucleic acid detection that is achieved by use of nucleic acid hybridization probes. The nucleic acid hybridization probes find use in detecting nucleic acid sequence targets in solution or bound to immobilized supports. Examples of applications where the composition and methods can be used to detect nucleic acid sequence targets in solution include PCR, real-time PCR, quantitative PCR, PNA clamp-mediated PCR and digital PCR. Examples of applications where the compositions and methods can be used to detect nucleic acid sequence targets immobilized to solid supports include northern blots, southern blots, dot blots, slot blots, microarrays, particle-based assays, In situ hybridization assays (ISH) such as, for example, chromagen in situ hybridization (CISH), RNA in situ hybridization (RISH), rapid FISH, Silver In Situ Hybridization (SISH), and FISH assays. Such applications are amenable to numerous fields, including medical diagnostics, molecular medicine, forensic science, specimen and organism cataloging, and microbial pathogen epidemiology.

The present invention is not limited to a particular target. The compositions and methods described herein find use in the detection of a variety of target nucleic acids (e.g., human or mammalian genomic nucleic acids), bacterial, viral, etc.

Probes may also be employed as isolated nucleic acids immobilized on a solid surface (e.g., nitrocellulose), as in aCGH. In some embodiments, the probes may be members of an array of nucleic acids as described, for instance, in WO 96/17958, which is hereby incorporated by reference it its entirety and specifically for its description of array CGH. Techniques capable of producing high density arrays are well-known (see, e.g., Fodor et al. Science 767-773 (1991) and U.S. Pat. No. 5,143,854), both of which are hereby incorporated by reference for this description.

A detailed description of the utility of the nucleic acid hybridization probes is presented below for FISH applications, although the probes find use in other applications.

FISH and other in situ hybridization methods may be performed on a variety of sample types. Example, include, but are not limited to formalin-fixed paraffin embedded (FFPE) tissue), fresh tissue, frozen tissue, cells (e.g., eukaryotic or prokaryotic cells); prepared using any suitable fixative. In some embodiments, touch prep or brushing (See e.g., Smoczynski et al., Gastrointest Endosc. 2012 January; 75(1):65-73) is utilized.

Touch prep specimens are generated by smearing or pressing onto a slide, applying pressure to the tissue, and fixing in ethanol under cool temperatures. In a specific embodiment the tissue is extracted surgically and smeared onto a glass slide by applying relatively weak pressure to tumor tissue and relatively strong pressure to normal tissue, followed by fixing in about 100% ethanol for approximately 10 minutes at about 4° C. In another specific embodiment, the samples to be analyzed by methods of the present invention are originally frozen in liquid nitrogen and stored at about −80° C.

For a typical ISH application, the following represents a typical procedure. Cells of a specimen are harvested, washed and pelleted. The cells of the pellet are usually washed in phosphate-buffered saline (PBS). The cells are suspended in PBS and re-collected by centrifugation. The cells can be fixed, for example, in acid alcohol solutions, acid acetone solutions, or aldehydes such as formaldehyde, paraformaldehyde, and glutaraldehyde. For example, a fixative containing methanol and glacial acetic acid in a 3:1 ratio, respectively, can be used as a fixative. A neutral buffered formalin solution also can be used, and includes approximately 1% to 10% of 37-40% formaldehyde in an aqueous solution of sodium phosphate. Slides containing the cells can be prepared by removing a majority of the fixative, leaving the concentrated cells suspended in only a portion of the solution.

The cell suspension is applied to slides such that the cells do not overlap on the slide. Cell density can be measured by a light or phase contrast microscope. The density of cells in these wells is then assessed with a phase contrast microscope. If the well containing the greatest volume of cell suspension does not have enough cells, the cell suspension is concentrated and placed in another well.

Prior to in situ hybridization, chromosomal probes and chromosomal DNA contained within the cell each are denatured. Denaturation process is performed in several ways. For example, denaturation can be effected with buffered solutions having elevated pH, with elevated temperatures (for example, temperatures from about 70° C. to about 95° C.), or with organic solvents such as formamide, ethylene carbonate, and tetraalkylammonium halides, or combinations thereof. For example, chromosomal DNA can be denatured by a combination of temperatures above 70° C. (for example, about 73° C.) and a denaturation buffer containing 70% formamide and 2×SSC (0.3M sodium chloride and 0.03 M sodium citrate). Denaturation conditions typically are established such that cell morphology is preserved. Chromosomal probes can be denatured by heat. For example, probes can be heated to about 73° C. for about five minutes.

After removal of denaturing chemicals or conditions, probes are annealed to the chromosomal DNA under hybridizing conditions. “Hybridizing conditions” are conditions that facilitate annealing between a probe and nucleic acid sequence target. Hybridization conditions vary, depending on the concentrations, base compositions, complexities, and lengths of the probes, as well as salt concentrations, temperatures, and length of incubation. The greater the concentration of probe, the greater the probability of forming a hybrid. For example, in situ hybridizations are typically performed in hybridization buffer containing 1-2×SSC, 50% formamide and blocking DNA to suppress non-specific hybridization. In general, hybridization conditions, as described above, include temperatures of about 25° C. to about 55° C., and incubation lengths of about 0.5 hours to about 96 hours. More particularly, hybridization can be performed at about 37° C. to about 40° C. for about 2 to about 16 hours.

Non-specific binding of chromosomal probes to DNA outside of the target region can be removed by a series of washes. Temperature and concentration of salt in each wash depend on the desired stringency. For example, for high stringency conditions, washes can be carried out at about 65° C. to about 80° C., using 0.2×SSC to about 2×SSC, and about 0.1% to about 1% of a non-ionic detergent such as Nonidet P-40 (NP40) or other suitable surfactant. Stringency can be lowered by decreasing the temperature of the washes or by increasing the concentration of salt in the washes.

Slides containing the samples are typically incubated in 2×SSC at 37° C. for 10-30 min. The slides are then incubated in 0.2 mg/ml pepsin at 37° C. for 20 min. Slides are subsequently washed twice in PBS at room temperature for 2 min. Cells are fixed in 2.5% Neutral Buffered Formalin at room temperature for 5 min. Slides are subsequently washed twice in PBS at room temperature for 2 min. The slides are subjected to dehydration by successive contact in solutions of 70%, 85%, and 100% ethanol at room temperature for 1 min. The slides are used immediately thereafter or stored at room temperature in the dark.

Hybridization can be performed with the HYBrite method or a conventional method. In the HYBrite method, a HYBrite™ system from Abbott Molecular (Downers Grove, Ill.) is used.

Conditions for specifically hybridizing the probes to their nucleic acid targets generally include the combinations of conditions that are employable in a given hybridization procedure to produce specific hybrids, the conditions of which may easily be determined by one of skill in the art. Such conditions typically involve controlled temperature, liquid phase, and contact between a chromosomal probe and a target. Hybridization conditions vary depending upon many factors including probe concentration, target length, target and probe G-C content, solvent composition, temperature, and duration of incubation. At least one denaturation step may precede contact of the probes with the targets. Alternatively, both the probe and nucleic acid target may be subjected to denaturing conditions together while in contact with one another, or with subsequent contact of the probe with the biological sample. Hybridization may be achieved with subsequent incubation of the probe/sample in, for example, a liquid phase of about a 50:50 volume ratio mixture of 2-4×SSC and formamide, at a temperature in the range of about 25 to about 55° C. for a time that is illustratively in the range of about 0.5 to about 96 hours, or more preferably at a temperature of about 32 to about 40° C. for a time in the range of about 2 to about 16 hours. In order to increase specificity, use of a blocking agent such as unlabeled blocking nucleic acid as described in U.S. Pat. No. 5,756,696 (the contents of which are herein incorporated by reference in their entirety, and specifically for the description of the use of blocking nucleic acid), may be used in conjunction with the methods of the present invention. Other conditions may be readily employed for specifically hybridizing the probes to their nucleic acid targets present in the sample, as would be readily apparent to one of skill in the art.

Upon completion of a suitable incubation period, non-specific binding of chromosomal probes to sample DNA may be removed by a series of washes. Temperature and salt concentrations are suitably chosen for a desired stringency. The level of stringency required depends on the complexity of a specific probe sequence in relation to the genomic sequence, and may be determined by systematically hybridizing probes to samples of known genetic composition. In general, high stringency washes may be carried out at a temperature in the range of about 65 to about 80° C. with about 0.2× to about 2×SSC and about 0.1% to about 1% of a non-ionic detergent such as Nonidet P-40 (NP40). If lower stringency washes are required, the washes may be carried out at a lower temperature with an increased concentration of salt.

Chromosomal probes can be directly labeled with a detectable label. Examples of detectable labels include fluorophores, e.g., organic molecules that fluoresce after absorbing light, and radioactive isotopes, e.g., ³²P, and ³H. Fluorophores can be directly labeled following covalent attachment to a nucleotide by incorporating the labeled nucleotide into the probe with standard techniques such as nick translation, random priming, and PCR labeling. Alternatively, deoxycytidine nucleotides within the probe can be transaminated with a linker. The fluorophore can then be covalently attached to the transaminated deoxycytidine nucleotides. See, e.g., U.S. Pat. No. 5,491,224 to Bittner, et al., which is incorporated herein by reference. Useful probe labeling techniques are described in Molecular Cytogenetics: Protocols and Applications, Y.-S. Fan, Ed., Chap. 2, “Labeling Fluorescence In Situ Hybridization Probes for Genomic Targets”, L. Morrison et. al., p. 21-40, Humana Press, © 2002 (hereafter cited as “Morrison-2002”), incorporated herein by reference.

Attachment of fluorophores to nucleic acid probes is well known in the art and may be accomplished by any available means. Fluorophores can be covalently attached to a particular nucleotide, for example, and the labeled nucleotide incorporated into the probe using standard techniques such as nick translation, random priming, PCR labeling, and the like. Alternatively, the fluorophore can be covalently attached via a linker to the deoxycytidine nucleotides of the probe that have been transaminated. Methods for labeling probes are described in U.S. Pat. No. 5,491,224 and Molecular Cytogenetics: Protocols and Applications (2002), Y.-S. Fan, Ed., Chapter 2, “Labeling Fluorescence In Situ Hybridization Probes for Genomic Targets,” L. Morrison et al., p. 21-40, Humana Press, both of which are herein incorporated by reference for their descriptions of labeling probes.

Exemplary fluorophores that can be used for labeling probes include TEXAS RED (Molecular Probes, Inc., Eugene, Oreg.), CASCADE blue aectylazide (Molecular Probes, Inc., Eugene, Oreg.), SPECTRUMORANGE™ (Abbott Molecular, Des Plaines, Ill.) and SPECTRUMGOLD™ (Abbott Molecular).

Additional examples of fluorophores that can be used in the methods described herein are: 7-amino-4-methylcoumarin-3-acetic acid (AMCA); 5-(and -6)-carboxy-X-rhodamine, lissamine rhodamine B, 5-(and -6)-carboxyfluorescein; fluorescein-5-isothiocyanate (FITC); 7-diethylaminocoumarin-3-carboxylic acid, tetramethyl-rhodamine-5-(and -6)-isothiocyanate; 5-(and -6)-carboxytetramethylrhodamine; 7-hydroxy-coumarin-3-carboxylic acid; 6-[fluorescein 5-(and -6)-carboxamido]hexanoic acid; N-(4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a diaza-3-indacenepropionic acid; eosin-5-isothiocyanate; erythrosine-5-isothiocyanate; 5-(and -6)-carboxyrhodamine 6G; and Cascades blue aectylazide (Molecular Probes, Inc., Eugene, Oreg.).

Probes can be viewed with a fluorescence microscope and an appropriate filter for each fluorophore, or by using dual or triple band-pass filter sets to observe multiple fluorophores. See, e.g., U.S. Pat. No. 5,776,688 to Bittner, et al., which is incorporated herein by reference. Any suitable microscopic imaging method can be used to visualize the hybridized probes, including automated digital imaging systems, such as those available from MetaSystems or Applied Imaging. Alternatively, techniques such as flow cytometry can be used to examine the hybridization pattern of the chromosomal probes.

Probes can also be labeled indirectly, e.g., with biotin or digoxygenin by means well known in the art. However, secondary detection molecules or further processing are then used to visualize the labeled probes. For example, a probe labeled with biotin can be detected by avidin conjugated to a detectable marker, e.g., a fluorophore. Additionally, avidin can be conjugated to an enzymatic marker such as alkaline phosphatase or horseradish peroxidase. Such enzymatic markers can be detected in standard colorimetric reactions using a substrate for the enzyme. Substrates for alkaline phosphatase include 5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium. Diaminobenzoate can be used as a substrate for horseradish peroxidase. Fluorescence detection of a hybridized biotin or other indirect labeled probe can be achieved by use of the commercially available tyramide amplification system.

One of skill in the art will recognize that other agents or dyes can be used in lieu of fluorophores as label-containing moieties. Suitable labels that can be attached to probes include, but are not limited to, radioisotopes, fluorophores, chromophores, mass labels, electron dense particles, magnetic particles, spin labels, molecules that emit luminescence, electrochemically active molecules, enzymes, cofactors, and enzyme substrates. Luminescent agents include, for example, radioluminescent, chemiluminescent, bioluminescent, and phosphorescent label containing moieties. Alternatively, detection moieties that are visualized by indirect means can be used. For example, probes can be labeled with biotin or digoxygenin using routine methods known in the art, and then further processed for detection. Visualization of a biotin-containing probe can be achieved via subsequent binding of avidin conjugated to a detectable marker. The detectable marker may be a fluorophore, in which case visualization and discrimination of probes may be achieved as described above for ISH.

In some embodiments, probes are designed to have labels placed at a common interval throughout the nucleic acid (e.g., one label group every 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12).

In some embodiments, a probe library comprises probes with different detectable labels (e.g., different colors of fluorescent signal).

Probes hybridized to target regions may alternatively be visualized by enzymatic reactions of label moieties with suitable substrates for the production of insoluble color products. A biotin-containing probe within a set may be detected via subsequent incubation with avidin conjugated to alkaline phosphatase (AP) or horseradish peroxidase (HRP) and a suitable substrate. 5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium (NBT) serve as substrates for alkaline phosphatase, while diaminobenzidine serves as a substrate for HRP.

In embodiments where fluorophore-labeled probes or probe compositions are used, the detection method can involve fluorescence microscopy, flow cytometry, or other means for determining probe hybridization. Any suitable microscopic imaging method may be used in conjunction with the methods of the present invention for observing multiple fluorophores. In the case where fluorescence microscopy is employed, hybridized samples may be viewed under light suitable for excitation of each fluorophore and with the use of an appropriate filter or filters. Automated digital imaging systems such as the MetaSystems, BioView or Applied Imaging systems may alternatively be used.

In array CGH, the probes are not labeled, but rather are immobilized at distinct locations on a substrate, as described in WO 96/17958. In this context, the probes are often referred to as the “target nucleic acids.” The sample nucleic acids are typically labeled to allow detection of hybridization complexes. The sample nucleic acids used in the hybridization may be detectably labeled prior to the hybridization reaction. Alternatively, a detectable label may be selected which binds to the hybridization product. In dual- or multi-color aCGH, the target nucleic acid array is hybridized to two or more collections of differently labeled nucleic acids, either simultaneously or serially. For example, sample nucleic acids and reference nucleic acids are each labeled with a separate and distinguishable label. Differences in intensity of each signal at each target nucleic acid spot can be detected as an indication of a copy number difference. Although any suitable detectable label can be employed for aCGH, fluorescent labels are typically the most convenient.

Exemplary methods of visualizing signals are described in WO 93/18186, which is hereby incorporated by reference for this description. To facilitate the display of results and to improve the sensitivity of detecting small differences in fluorescence intensity, a digital image analysis system can be used. An exemplary system is QUIPS (an acronym for quantitative image processing system), which is an automated image analysis system based on a standard fluorescence microscope equipped with an automated stage, focus control and filterwheel (Ludl Electronic Products, Ltd., Hawthorne, N.Y.). The filterwheel is mounted in the fluorescence excitation path of the microscope for selection of the excitation wavelength. Special filters (Chroma Technology, Brattleboro, Vt.) in the dichroic block allow excitation of the multiple dyes without image registration shift. The microscope has two camera ports, one of which has an intensified CCD camera (Quantex Corp., Sunnyvale, Calif.) for sensitive high-speed video image display which is used for finding interesting areas on a slide as well as for focusing. The other camera port has a cooled CCD camera (model 200 by Photometrics Ltd., Tucson, Ariz.) which is used for the actual image acquisition at high resolution and sensitivity. The cooled CCD camera is interfaced to a SUN 4/330 workstation (SUN Microsystems, Inc., Mountain View, Calif.) through a VME bus. The entire acquisition of multicolor images is controlled using an image processing software package SCIL-Image (Delft Centre for Image Processing, Delft, Netherlands).

In some embodiments, the present disclosure provides kits and systems for the amplification and/or analysis of nucleic acids. In some embodiments, kits include reagents necessary, sufficient or useful for analysis and detection of copy number or gene expression changes (e.g., primers, probes, anchors, solid supports, reagents, controls, instructions, etc.). For example, in some embodiments, kits comprise primers and anchors for amplification and sequencing of regions of interest and control regions. In some embodiments, kits include analysis software (e.g., to analyze sequencing data).

In some embodiments, kits comprise one or more containers that comprise reagents, primers, probes, anchors, solid supports, buffers, and the like. In some embodiments, each component of the kit is packaged in a separate container. In some embodiments, the containers are packed and/or shipped in the same kit or box for use together. In some embodiments, one or more components of the kit are shipped and/or packaged separately.

The assays and kits of the can be adapted or optimized for point of care assay systems, including Abbott's Point of Care (i-STAT™) electrochemical immunoassay system Immunosensors and methods of manufacturing and operating them in single-use test devices are described, for example in U.S. Pat. No. 5,063,081 and published U.S. Patent Application Nos. 20030170881, 20040018577, 20050054078, and 20060160164 (incorporated by reference herein for their teachings regarding same).

In some embodiments, systems include automated sample and reagent handling devices (e.g., robotics).

EXPERIMENTAL
Example 1
Generation of Probes

UCSC Genome browser was used to identify sequence in the region of the HER2 locus with coordinates hg18_dna range chr17:35004678-35230380. This sequence was downloaded, coded such that regions identified by RepeatMasker were masked as lower case. The 225703 base sequence was treated with a software program that removed all sequence with more than 3 consecutive lower case letters, returning the stretches of repeat-free sequence along with their positions in the original sequence (FIG. 2).

These sequences ranged in size from very small to 5615 bases. Only the longest fragments are selected for generating sequence library. A minimum length cutoff of 1200 bp yielded 35 sequences containing 76904 bp of sequence. PCR amplification of each individual sequence is expected to give variable yields of subdomains of sequence depending on the length of the amplicon, therefore sequences were divided into multiple overlapping subsequence, 1200 bp each. All subfragments were designed to include at least 100 bp overlap with neighboring subfragments to accommodate variations in primer positions. This process generated 95 candidate sequences.

For each sequence a primer pair was designed with the web based program “BatchPrimer3” seeking generic primers to generate amplicons with Min 800, Opt 1200, Max 1200; primer length min 22, opt 25, max 30; Tm min 65, opt 70, max 75. Primer sequences were downloaded as an xls file and copied into the spreadsheet for adjusting format to make it suitable for placing the order.

Primers were ordered from IDT as Mixed Forward and Reverse primers 12 nmole each dried in deep-well plate. To each well, 240 uL water (5 Prime 2900132) was added to give 50 uM each. Dilutions were prepared as needed for PCR.

PCR of genomic DNA yielded clean 1200mer product for only about half of the wells, therefore a preparation of BAC DNA containing the desired locus was used to generate a much higher success rate. BAC clones in E. coli covering the locus were obtained from Genome Systems, Inc. St. Louis, cultures grown and DNA isolated by “mini-prep.”

PCR Master Mix: A PCR kit containing Phire Hot Start II DNA Polymerase (Thermo F-122L) was used. It contained 400 uL 5× reaction buffer+40 uL 25 mM dNTP (Roche Diagnostics, Indianapolis, Ind.)+40 uL Phire polymerase+60 uL DMSO+1260 uL water+0.5 ug BAC DNA template.

PCR: To wells of a 96 well plate was added 2 uL each primer pair 5 uM+18 uL Master Mix; capped wells, placed on thermocycler with program 98 deg 30 sec, 30× (98 deg 8 sec, 72 deg 30 sec, 72 deg 2 min), 72 deg 10 min, 4 deg. At completion 5 uL of each reaction was sampled into 20 uL water with trace 6×DNA Loading dye (Thermo R0611), 20 uL was transferred to wells of an eGel 96 agarose gel (Life Technologies), and electrophoresed was 8 min. 94 of 96 wells show clean bands at expected MW (FIG. 3).

Extend PCR to maximize yield. To each well containing the remaining 5 uL from electrophoresis sample, 10 uL of the same 5 uM primers+10 uL water+25 uL DreamTaq Green 2×pcrMM was added, and the tube was capped and placed on a thermocycler with program 10× (95 deg 30 sec, 55 deg 30 sec, 72 deg 2 min) 72 deg 6 min. At completion, to each well 6 uL of (30 uL 1M MgCl2+192 uL 25 mM dNTP+378 uL water+100 uL DreamTaq Green 2×pcrMM) was added and the tube was capped and placed on thermocycler with program 6× (95 deg 30 sec, 55 deg 30 sec, 72 deg 2 min) 72 deg 10 min.

The contents of columns were combined to give 6 amplicon mixtures representing different loci of the genomic range, and the DNA isolated by precipitation with isopropanol and resuspended in water, yielding 100 uL solutions containing 52-75 ug DNA.

Fragmentation by sonication: Approx 180 ug DNA 1200-mers from PCR wells 1-48 and 49-96 were combined in tubes with water to give 400 uL, with sodium acetate at 300 mM. The tubs were placed in beaker of ice water in a sonicator cabinet, sonicated (sonicator Branson 450) output control 3, 30% duty cycle 16 min. The sonication products were precipitated with isopropanol and resuspended in 200 uL 600 mM NaCl in 20 mM Tris pH 8.2. The samples were fractionated by HPLC with MonoQ column (GE Life Sciences), BufferA=20 mM Tris pH 8.2; Buffer B=A+2.0M NaCl, 0.4 mL/min % B=40-50 over 32 min, collecting fractions. DNA elutes in a broad peak centered at about 15 min. Fractions were combined to give 7 pools for each. Electrophoresis on a 3% Agarose gel visualized with Ethidium Bromide shows fractions (labeled H15, H18, H21, H24, H27, H30, H33) ranging from about 80 bp to about 400 bp (FIG. 4).

Amination of fragmented DNA: The pooled fractions were concentrated by isopropanol precipitation, resuspended in 20 uL water and denatured by heating 1 min in boiling water. To the 20 uL denatured DNA was added 180 uL of a mixture of 1000 uL water, 600 uL trifluoroacetic acid (Sigma, St Louis Mo.) 348 uL ethylenediamine and 190 mg sodium metabisulfite, and the mixture incubated 20 min at 65 deg. The mixtures were then desalted by sephadex G25 and the desalted product concentrated by isopropanol precipitation and resuspension in water.

The aminated products were labeled with carboxytetramethylrhodamine using its NHS ester (Life Technologies C1171) by means well known in the art. Labeled product was isolated from residual unbound dye by ultrafiltration using 10 kDa filters (Nanosep 10 Omega, Pall Corporation, Ann Arbor, Mich.), followed by filtration through 0.22 u filters (Millipore UFC30GV00, Billerica, Mass.).

Preparation of Probes Via Adaptor Mediated PCR

Fragments of the desired size were also modified by ligating adaptors, and using the product as a template for PCR using primers corresponding to the adaptor sequence. This provides an efficient means of producing larger quantities of product of the desired size, as well as providing a means of coupling additional functional groups to the product. The process consists of modifying the fragments to give 5′ phosphorylated, blunted ends, followed by ligating adaptors of the desired sequence. The adaptors can be designed to contain restriction sites such that the dsDNA PCR product can be cleaved with appropriate restriction enzymes either to remove unwanted adaptor sequence or to reveal sticky ends suitable for ligating additional groups.

End repair of the fragments was by using the Thermo Fast DNA End Repair Kit K0771 according to directions, starting with 4 ug each fraction H18, H24, H30. Blunted products were isolated using spin columns (Invitrogen K310001) Treatment of 4 ug sonicated, fractionated DNA yielded approx 3.5 ug blunted product H18b, H24b, H30b, with respective concentrations 74, 66, 70 ng/uL.

An adaptor mix was prepared by combining each oligonucleotide G6a, G6b, Gc6a, Gc6b, BsPD, BsTB, BsPDc, BsTBc with 20 mM Tris pH 8.0 and 250 mM NaCl, the mixture heated 1 min in boiling water and cooled to room temperature. T4 DNA Ligase (Invitrogen A13726, a kit containing the ligase at 5 Units/uL, and reaction buffer) was used to ligate the adaptors to each blunted DNA fraction: to 10 uL blunted DNA fraction was added 5 uL adaptor mix, 2 uL 10× Ligase buffer, 2 uL 50% PEG4000 and 1 uL, and the mixture left overnight at room temp. Adapted products were isolated using spin columns (Invitrogen K310001) eluting in 50 uL elution buffer, and labeled H18T, H24T, H30T with respective concentrations 37, 39, 30 ng/uL.

PCR of adapted HER2 fractions was performed at 20 uL scale each using 1 uL of the adapted template H18T, H25T, H30T, and a commercial PCR Master Mix (DreamTaq Green Thermo K1081). Each reaction contained a single primer G6a or BsPD at 5 or 10 uM. Cycling conditions were 24× (95 deg 30 sec, 52 deg 30 sec, 72 deg 30 sec). Products were denatured by adding 1 uL 1M sodium hydroxide to 4 uL product, and analyzed on 3% Agarose/EtBr gel. For all primers and primer concentrations tested PCR products show bands corresponding to the template sizes (FIG. 5).

Products from the PCR shown in FIG. 5 were used as template for amplification using 10 uM BsPD24 primer; 10 uL of BsPD 10-18, BsPD 10-24 and BsPD 5-30 were added to give 800 uL in final 1× Master Mix. These were split to 8×100 uL portions and amplified with cycling conditions 12× (95 deg 30 sec, 60 deg 30 sec, 72 deg 2 min). An additional volume of 2.1 uL of a mixture of 41.5 uL 25 mM dNTP (Roche Diagnostics, Indianapolis, Ind.) and 13 uL 1.0M magnesium chloride (Sigma, St Louis Mo.) were added to each well and the mixtures subjected to an additional 8× (95 deg 30 sec, 60 deg 30 sec, 72 deg 4 min). The combined products were precipitated with isopropanol, resuspended in 200 uL water each and further precipitated with polyethylene glycol to separate PCR product from primers. Final PCR products were dissolved in 200 uL water, labeled H18B, H24B, H30B, concentrations measured at 1126, 1270, 1357 ng/uL.

Restriction digestion of PCR products to decrease adaptor portion: Restriction enzyme BspDI (New England Biolabs Ipswich Mass.) was used. To 260 uL water, 40 uL 10× CutSmart buffer BspDI (New England Biolabs Ipswich Mass.), 100 uL of the above PCR products H18B, H24B, H30B, and 10 uL 10 U/uL BspDI was added, and the mixtures were incubated for 16h at 37 deg. Reactions were labeled H18R, H24R, H30R.

After a 16h incubation, samples of each were taken for electrophoresis, comparing digested with undigested products. To wells of a PCR strip were added 8 uL 2× loading dye (Thermo Fisher Scientific), 0.6 uL (H18B, H24B, H30B), 2.4 uL (H18R, H24R, H30R)+water to 10 uL; Each mixture was split to 2×5 uL each, to all added 4 uL water. To one of each added 1 uL 1M NaOH and heated 95 deg 30 sec to denature. Five μL was loaded each to well of 3% Agarose/EtBr gel and electrophoresed. In the gel image “−” and “+” correspond to absence and presence of NaOH. All digestions show the smaller size and presence of end fragments expected for successful cleavage by BspDI (doublet probably from annealing fragment with residual primer). Denaturation by NaOH shows further decrease in size for both undigested and digested products. Higher MW smears from extended annealing of fragments are eliminated by denaturation.

The products H18B, H24B, H30B, H18R, H24R, H30R were precipitated with isopropanol, resuspended in 20 uL water and subjected to amination and labeling with carboxytetramethylrhodamine by the procedure described above.

FISH Hybridization Conditions

The targeting probes were hybridized to human chromosomal DNA in lymphocytes bound to glass microscope slides. In a typical experiment the reagent mixture consists of 7 μL of LS/WCP hybridization buffer (Abbott Molecular (Des Plaines, Ill.)) and 3 μL water containing 2000 ng sonicated human placental DNA, 500 ng COT-1 DNA (Life Technologies™ (Grand Island, N.Y.)), 50 ng of probe CEP17-SG buffer (Abbott Molecular (Des Plaines, Ill.)) and 100 ng of test probe.

The microscope slide was dehydrated by successive immersion in 70%, 85% and 100% ethanol, then air dried. The test solution (10 μL) was placed on the slide and covered with a 22×22 mm slip, causing the solution to spread over the covered area. Rubber cement was applied to seal the edges and the slide placed in an instrument that controls the temperature. The temperature was raised to 70° C. for 5 min. to denature the DNA of both the sample and the reagent, then lowered to 45° C. 1 h to allow time for the reagent to hybridize to its target. After completion of the hybridization time, the rubber cement and slips were removed, and the slide washed 2 min. in a solution of 0.4×SSC and 0.3% NP40 at 73° C., then 1 min in 2×SSC, 0.1% NP40 at room temperature, then air dried.

The slide was prepared for viewing by placing 10 uL of a solution of DAPI on the target area and covering with a slip. The slide was viewed with a fluorescence microscope equipped with filters suitable for the fluorophore of interest.

Fluorescence Microscopy of Resultant Hybridization Patterns

Ten μL of DAPI-II (Abbott Molecular (Des Plaines, Ill.)) was placed on the slide at the position of the target, covered with a 22×22 mm slip and viewed under fluorescence microscope equipped with filters that allow simultaneous visualization of DAPI, fluorescein (green) and TAMRA (orange) signals. The photograph (FIG. 7) shows the pattern of orange and green signals consistent with those expected for the HER2 locus (orange) stained by the test probe and the centromeric position stained by CEP17-SG (green). On Metaphase chromosomes the orange and green signals are at adjacent positions on the same chromosome, while interphase nuclei show two strong signals for each, at arbitrary distances. Similar results were seen for probes prepared via Adaptor Mediated PCR of the same HPLC fractions. Removal of the adaptor ends by restriction digestion prior to labeling yielded probe with indistinguishable performance characteristics.

Primer Sequences Used for 1200mer Fragments:

SEQ

Well-

ID

Position
Name
Sequence
NO

A1
HER2_P_F_01
AAAGGGGCCAGTTATGCAG
1

A1
HER2_P_R_01
GGTCCGTGGAATTGGATTATT
2

B1
HER2_P_F_02
CTGGCGAAGGGGATCTATTT
3

B1
HER2_P_R_02
CATCTGTGTGCGGAATGACT
4

C1
HER2_P_F_03
TTGTCTGGAAGACGCAGAAC
5

C1
HER2_P_R_03
GTGCCAGTCTGTGCCACTC
6

D1
HER2_P_F_04
GGAGTGAGCTGGTTGGTCAC
7

D1
HER2_P_R_04
ATCTGTTCTCGCCAGAGTCG
8

E1
HER2_P_F_05
GGTTTTGCTTTGGCTCTTTG
9

E1
HER2_P_R_05
GACGTGAGAAGAAGGCCAAG
10

F1
HER2_P_F_06
GACCTGACCTAGCAGCCTTG
11

F1
HER2_P_R_06
TCTCTTGGCAACCTTTGCTT
12

G1
HER2_P_F_07
TCACCCAGGGAAACTTTGTC
13

G1
HER2_P_R_07
CCCAGAGATGCTCCAAGAAC
14

H1
HER2_P_F_08
AAAAGAGAAGCAGGCACAGC
15

H1
HER2_P_R_08
ACTTATCCCGAGGCCTGATT
16

A2
HER2_P_F_09
CTGCTCATCACACCATCTCG
17

A2
HER2_P_R_09
CTCCCTGACAAGCAGGAAAG
18

B2
HER2_P_F_10
GGGGTATGTGCCTTGCTCTA
19

B2
HER2_P_R_10
TGACATGTTTAGGGGTGTGG
20

C2
HER2_P_F_11
GGCCAACTCCTTTCTTCCAT
21

C2
HER2_P_R_11
TTGTATTTCGGTGGACTCAGG
22

D2
HER2_P_F_12
TTTCTCTCCCTGCCTTCTCC
23

D2
HER2_P_R_12
GACACCTGGGTTTGATCCAC
24

E2
HER2_P_F_13
GGTTCCCATGGAGACATAGC
25

E2
HER2_P_R_13
GACCTCGGTCTCTCAGCATC
26

F2
HER2_P_F_14
CCGCTCCACCAAAATACATA
27

F2
HER2_P_R_14
GCATTTGACCAAAGGGAAAC
28

G2
HER2_P_F_15
GAGTCCTGCCCCATGCTC
29

G2
HER2_P_R_15
AATACCGGCTCAGGACAGG
30

H2
HER2_P_F_16
CCCCCTCACATCTGACAATC
31

H2
HER2_P_R_16
TGCTGAACAGTTCTCCAGCTAA
32

A3
HER2_P_F_17
ACACGCTGAGCCAGATTGAC
33

A3
HER2_P_R_17
GGCACAAACGAGTACAGCAG
34

B3
HER2_P_F_18
CCCTCCCCATGTGAATTTT
35

B3
HER2_P_R_18
TGGGCTCCTCTCTTTCTCTG
36

C3
HER2_P_F_19
CTTGGCCCCAGGATTTAGA
37

C3
HER2_P_R_19
GGAGACAGGTGTGAGCCTCT
38

D3
HER2_P_F_20
TCCCACTCCTATGAGCAACA
39

D3
HER2_P_R_20
TGGAGTCCAGCAGAGAGGAT
40

E3
HER2_P_F_21
GAAGCATTCAGACCCTCTGC
41

E3
HER2_P_R_21
GCGTGTGTCTCTGCCTCTG
42

F3
HER2_P_F_22
CGAGACGCAGAGACACTCAG
43

F3
HER2_P_R_22
CTGCATCTCAGCTCAGCAAC
44

G3
HER2_P_F_23
GCAAGAGAGTTCCTGGCAGT
45

G3
HER2_P_R_23
GCCCTTCCTCTCTCCAGTTT
46

H3
HER2_P_F_24
GCAAGAGAGTTCCTGGCAGT
47

H3
HER2_P_R_24
GCCCTTCCTCTCTCCAGTTT
48

A4
HER2_P_F_25
AGCACTGTTTGTTCCCTGCT
49

A4
HER2_P_R_25
CTCGGGACTCCTGTGTTTTG
50

B4
HER2_P_F_26
AAAGCTGGAGACTGGGGAGT
51

B4
HER2_P_R_26
GTGCAGATCTGTGCAAATGG
52

C4
HER2_P_F_27
AATTTAACAGGCAGGCAAGG
53

C4
HER2_P_R_27
GTAAGGCAGGAGAGCAGGTG
54

D4
HER2_P_F_28
AAGACCTGGCTCTTGACTGC
55

D4
HER2_P_R_28
CCAAAAGATGGAAAGGAGCA
56

E4
HER2_P_F_29
GGCTCAAGATGAAGCTCTGC
57

E4
HER2_P_R_29
GGCAGAGAATACCCCCTCA
58

F4
HER2_P_F_30
AGCATAGCACCCTGCTCACT
59

F4
HER2_P_R_30
TCTGAGGCCTGGTTCTCATT
60

G4
HER2_P_F_31
CCCTGGAAAGCTTAACCTCA
61

G4
HER2_P_R_31
GCTTCCCCTGAAAGAGGAGT
62

H4
HER2_P_F_32
GGAGGATTCCAAGTCACCAC
63

H4
HER2_P_R_32
GCCAAGAGTCATTGCTGGAG
64

A5
HER2_P_F_33
GTAATGGGGCGTCCTGATAG
65

A5
HER2_P_R_33
TGTCAGAGCGGTACGAAGAA
66

B5
HER2_P_F_34
AGGCTGGAAAGAGGAAGGAG
67

B5
HER2_P_R_34
CCTGCTCCAAGTTCTTACGG
68

C5
HER2_P_F_35
AGAGGAGAGGTGGCATCAGA
69

C5
HER2_P_R_35
AAGGATGGGAGCCGAGTCT
70

D5
HER2_P_F_36
AAGACCCCTGTGCAAGGTTA
71

D5
HER2_P_R_36
CAAGATACCCTGGAGGAGCA
72

E5
HER2_P_F_37
CTTAGCCCCTTGCAGCTCTA
73

E5
HER2_P_R_37
GGGATCTGGGCTGGTCTC
74

F5
HER2_P_F_38
CTGTTCTCCGGTGCTCTGTC
75

F5
HER2_P_R_38
GGGCATGTTGCTCTCTGTTT
76

G5
HER2_P_F_39
AGAGAGAGAACAGGCCACGA
77

G5
HER2_P_R_39
TCTTGTTCCACAGCACCATC
78

H5
HER2_P_F_40
AGAGAACAGGCCACGAACAT
79

H5
HER2_P_R_40
CCCATCTGTGCCTTAAGAGG
80

A6
HER2_P_F_41
GCCGTTGTAGGAGGATTCAA
81

A6
HER2_P_R_41
CAGAGCAATCTGGTCCTCCA
82

B6
HER2_P_F_42
CGTGTTTGCACCTTTGTCTG
83

B6
HER2_P_R_42
ATACAAAGGTCCCCCAGGAG
84

C6
HER2_P_F_43
AGGTGTTGGGGTAGAACTGG
85

C6
HER2_P_R_43
CCCTGCTGGTGGTAGGTCT
86

D6
HER2_P_F_44
GAGGTGTCGGAGGAGAACTG
87

D6
HER2_P_R_44
TATTGCGGCACTAACAGAGG
88

E6
HER2_P_F_45
AGCACAGAGAGGCTGAGAGG
89

E6
HER2_P_R_45
CCTTCCCCTCTGGATGAGTC
90

F6
HER2_P_F_46
CAGTCCTGGCTTCTGTGTCC
91

F6
HER2_P_R_46
TCCCTTAGAACTGCCACACA
92

G6
HER2_P_F_47
CTGTGTCTCGCTCCACACC
93

G6
HER2_P_R_47
CCCCTCCCATCTCTCTTCTC
94

H6
HER2_P_F_48
CAGACCAGAACGAGGGAGAG
95

H6
HER2_P_R_48
GTGGGCATGTGAGATGAGTG
96

A7
HER2_P_F_49
GAAACCAGACCCAGCCATAA
97

A7
HER2_P_R_49
CCAGCCTTGGAGTCTGTTCT
98

B7
HER2_P_F_50
GCCCTGAAAGGGAGTATGGT
99

B7
HER2_P_R_50
GATGATCCTGGGGTCAGAGA
100

C7
HER2_P_F_51
TTGAGGCACACAGCTCTGAC
101

C7
HER2_P_R_51
TCTGTGCCTCCACTGTCATC
102

D7
HER2_P_F_52
ATCCAGGACCCAGAAGAGC
103

D7
HER2_P_R_52
AGCGTCCCTAAAGCCTTGTT
104

E7
HER2_P_F_53
CCATACTCCTCCCAGTGCTC
105

E7
HER2_P_R_53
GGCCAGTTTTCCTGGTACAT
106

F7
HER2_P_F_54
GGAGGAGTAGAGGGCAGGAC
107

F7
HER2_P_R_54
CTCTTCTCACCTCCCCCTTC
108

G7
HER2_P_F_55
TCGTGACAACCAAAGGAACA
109

G7
HER2_P_R_55
CCTCCCAAATCTGAGGAAAG
110

H7
HER2_P_F_56
CCCCATTGTTGTTGTTTTCC
111

H7
HER2_P_R_56
GATTCCAGTTGTGGGCATCT
112

A8
HER2_P_F_57
CCAGGTGATTCATCTCACCA
113

A8
HER2_P_R_57
GGCAGGTAGGTGAGTTCCAG
114

B8
HER2_P_F_58
GTCTTGCCCTGAGGAGGTG
115

B8
HER2_P_R_58
TGATCATGCTGGCAAGAGAG
116

C8
HER2_P_F_59
AGCATCTGGACCTAGCATGG
117

C8
HER2_P_R_59
GCACAAAGCAGAGGCACATA
118

D8
HER2_P_F_60
CCTGCTGCCTCTTCTCTCAG
119

D8
HER2_P_R_60
CATGACCAGCTCTCAAAGCA
120

E8
HER2_P_F_61
GGCTTTGAAGCCCAGGAT
121

E8
HER2_P_R_61
GACCGCAGGGGACTTTTAG
122

F8
HER2_P_F_62
GCCCACCTTTCTCCCATAGT
123

F8
HER2_P_R_62
ACCCTAGCACAGCCACAGTC
124

G8
HER2_P_F_63
GCTGTGGTTTGTGATGGTTG
125

G8
HER2_P_R_63
GGGATCCCATCGTAAGGTTT
126

H8
HER2_P_F_64
AGGACCTGCTGAACTGGTGT
127

H8
HER2_P_R_64
CCTCAAGAGTGGCTTTGGAC
128

A9
HER2_P_F_65
CCAAAGGTTCTGGCTGAAGA
129

A9
HER2_P_R_65
GGCAACGTAGCCATCAGTCT
130

B9
HER2_P_F_66
CAGCTCATCTACCAGGGTCA
131

B9
HER2_P_R_66
CTTGATGCCAGCAGAAGTCA
132

C9
HER2_P_F_67
CTTCCCCTAATGGGTCACCT
133

C9
HER2_P_R_67
CTGGATGTCTGGCTCCTCAT
134

D9
HER2_P_F_68
CTCGTTGGAAGAGGAACAGC
135

D9
HER2_P_R_68
TAGAAGATTCCGTGGCCTTG
136

E9
HER2_P_F_69
CCCTTTGACGACCAGATCAT
137

E9
HER2_P_R_69
GCAATCGTGTAGGGTTGGAG
138

F9
HER2_P_F_70
TTCCTAAGGCCACTCACCAG
139

F9
HER2_P_R_70
GCCTGTGGGGAAAAACCTAT
140

G9
HER2_P_F_71
CCAGAGCTTTCTCCAGGTCA
141

G9
HER2_P_R_71
GAGACCCAGCCTTTCCCTAC
142

H9
HER2_P_F_72
CCGCCTCTGACTTCTCTGTC
143

H9
HER2_P_R_72
TGCATTCATTCTCTGTCCTCA
144

A10
HER2_P_F_73
CTCTCCTCCGACTTGGCTTT
145

A10
HER2_P_R_73
ATCAGTTTGTCCCCTCAACG
146

B10
HER2_P_F_74
ACTGCAGAGACACTCCAGCA
147

B10
HER2_P_R_74
CCCTGTGTGGATGAAGTTCC
148

C10
HER2_P_F_75
CTGGCCCTCTCTGATCTCTG
149

C10
HER2_P_R_75
TCACTTATAGGGGCTGCACA
150

D10
HER2_P_F_76
CTAACCCCTTCCAAGCACTG
151

D10
HER2_P_R_76
ATCCACCCATTTGTCTGAGG
152

E10
HER2_P_F_77
TCATTCTGTCCTTCCCCAAG
153

E10
HER2_P_R_77
GTTTTTCCGGAAGACGAAGC
154

F10
HER2_P_F_78
CCTCTGCCTGAGGAGGTAAA
155

F10
HER2_P_R_78
GCACAGGACTTAAGGGTGGA
156

G10
HER2_P_F_79
CAAGTCCTGCTCACTCATGC
157

G10
HER2_P_R_79
CCAGCACCTCAGGAAGGTAG
158

H10
HER2_P_F_80
ATCGAAGGCAGAAACACAGC
159

H10
HER2_P_R_80
CTGGTGAGGAGGACAGGTTG
160

A11
HER2_P_F_81
CCTCTCGACCTCAAGCTCTC
161

A11
HER2_P_R_81
CTTCTTGTGCAGGGAAAAGG
162

B11
HER2_P_F_82
CTGGACAGGTGGTGAAATGC
163

B11
HER2_P_R_82
GGGCTCTGGGAAGGAGTTAG
164

C11
HER2_P_F_83
GGGCTGGGACCTCAGATACT
165

C11
HER2_P_R_83
TTCGAGAACGCTTGTGGAG
166

D11
HER2_P_F_84
GCCCCACACATCTACTGGAG
167

D11
HER2_P_R_84
GCTTCACAGCTCCCTCCTC
168

E11
HER2_P_F_85
GTTGGGGTAGGGGAGGATAC
169

E11
HER2_P_R_85
CAGCTGCACTTCTGAGAAACA
170

F11
HER2_P_F_86
ACAGGCACACATGGAGACAG
171

F11
HER2_P_R_86
TTTCAGCCCTGGAGAGAAGA
172

G11
HER2_P_F_87
GGCCAGTGTTTCTGGTCTTC
173

G11
HER2_P_R_87
CCTGGTTCCACTGGTCCTTA
174

H11
HER2_P_F_88
GCCTAGCCCCACATTTGTTA
175

H11
HER2_P_R_88
ACACGTGTGGTCTGTGGATG
176

Al2
HER2_P_F_89
CGGGATGAAACCCTTCTACA
177

A12
HER2_P_R_89
TTTTAATGCACAAGGGCAGA
178

B12
HER2_P_F_90
CTTACAGGGTTCCAGCAAGG
179

B12
HER2_P_R_90
AGGGGTGGTGTGCATTATGT
180

C12
HER2_P_F_91
CCAGCATTTTGTGACCTCCT
181

C12
HER2_P_R_91
GCTGTGTTCACAGGGGTAGC
182

D12
HER2_P_F_92
TTTCACTGGAGATGGGAAGG
183

D12
HER2_P_R_92
TGTATCCCATAGCCCTCACC
184

E12
HER2_P_F_93
ATACCGATCATGGCTTCGAT
185

E12
HER2_P_R_93
AGTGTCCCCACTCTTTGCAG
186

F12
HER2_P_F_94
CGCTTCTCACCTGGAACAAG
187

F12
HER2_P_R_94
GGGCAGTGACAAATTTTGGA
188

G12
HER2_P_F_95
GTTAAGTTGCCTGCTGCTCA
189

G12
HER2_P_R_95
TGGAGACACCTCAAGAACAGG
190

Sequences Used in Adaptor Modification and Adaptor Mediated PCR

SEQ ID NO

BsPD
CTCTACCATCGATCACAGTG
191

BsTB
CTCTACCTTCGAAGAACGAC
192

BsPD24
CCAGCTCTACCATCGATCACAGTG
193

BsTB24
CCAGCTCTACCTTCGAAGAACGAC
194

BsPDc6
CACAGTG
195

BsTBc6
GAACGAC
196

BsPDc
CACTGTGATCGATGGTAGAG
197

BsTBc
GTCGTTCTTCGAAGGTAGAG
198

Gc6a
CACTTCTCTC
199

Gc6b
CACTCACATT
200

G6a
GGAGATGAGTGGATGGGAGAGAAGTG
201

G6b
GTGGTAGGAGGGATGAATGTGAGTG
202

Example 2

Designs were carried out using model targets p53, HER2 and p16 based on available BAC probes for comparison. Genomic sequence corresponding to the targets, with repeat sequence flagged for removal, was identified with UCSC Genome Browser.

For PCR probes, computer applications were used to isolate the repeat-free portions of the sequence, and to identify fragments to target for preparation as “kilomers”. The web based application “BatchPrimer3” was used to generate primer sequences to amplify as much of each kilomer sequence as possible, and the identified primer sequences were synthesized by IDT in a 96-well format. For Oligo and Oligo-PCR hybrid probes, the repeat-free portions of sequence were further processed to identify fragments of specified size and GC content. Excel spreadsheets were used for such processes as appending common adaptor sequences, sorting and formatting the sequence lists for placing synthesis orders.

Bulk DNA, which ends up in the final probe, was generated either by oligo synthesis, Oligo-PCR hybrid, or by 1- or 2-step PCR. The 1-step PCR method is as follows: After PCR using BAC or genomic DNA template the products are combined and fragmented by sonication to give a product that can be chemically labeled by the same process used for BAC based FISH probes. FISH probes made this way are structurally identical to the AM BAC probes—the only difference is that the PCR probes exclude the repeat sequence and vector sequence present in BAC DNA. The 2-step method is as follows: The sonicated product is ligated to adaptors to make a single template mixture containing all the chosen targeting sequence. This template can then be used in a single PCR reaction using a single set of primers to generate bulk DNA for amination and labeling. In this case sonication is no longer needed since the amplified product is already the desired size. The template is prepared once, stored indefinitely and samples taken for each new preparation. The adaptor sequences are present at the 5′ and 3′ ends of the product. While they can be removed by restriction digestion, testing shows that their presence does not damage the performance in FISH assays.

Amine groups for attachment of fluorophore labels were introduced either byinclusion of aminoallyl dUTP in the PCR reaction, or by chemical amination. The chemical amination was performed by the bisulfite/TFA/ethylenediamine process used with AM probes, but with the exception that a small amount of tetramethylethylenediamine was added after desalting the reaction mixture, but prior to ethanol precipitation. This displaces residual unlinked ethylenediamine that would otherwise compete for fluorophore in the labeling reaction.

Labeling the aminated DNA with fluorophore was done by a modification of an established process more suited to the numerous small scale reactions. In this modification the aminated DNA is combined with a reaction buffer of tetramethylethylenediamine and sodium chloride in 25% DMSO, the active fluorophore added and the mixture is incubated 2h at 60° C. The product is isolated by ethanol precipitation and subjected to 75° C. 72 h formamide treatment standard.

HER2 PCR Probes:

For HER2 PCR probes, three variants were generated, all using 76 kb of repeat-free sequence. For the simplest “1-step PCR” probe, the PCR generated DNA was treated the same as BAC DNA in conventional probes: fragmentation by sonication, followed by chemical amination and labeling. For the “2-step PCR” probe, the sonication fragments were ligated to adaptors to make a template. This template was amplified with a single primer to generate bulk DNA ready for amination and labeling. In the third format, “2-step PCR with aminoallyl dUTP”, this same template was amplified with a primer pair in the presence of aminoallyl dUTP to generate the aminated product, ready for labeling with any desired fluorophore.

Generation of Repeat-Free Sequence in HER2 Locus:

Sequence for the HER2 PCR format was identified using the locus defined by the P1 clones pVYS 174 C, E, H, I. These clones include 226 kb at hg18_dna range chr17:35004678-35230380. These coordinates were entered into UCSD Genome Browser, and the corresponding sequence presented using the Repeat Masker function to give the portions known to be repeat sequence in lower case. The upper case “unique” sequence portions were copied using an application to select out the upper case unique subsequences, retaining the position information of each. This yielded 35 sequences longer than 1200 bp (1234 to 5615 bp), totaling 76904 bp. These were processed with another application to break all the sequences into a total of 95 sequences, 1200 bp each, with at least 300 bp overlap. The 1200 bp sequences were entered into the web based application “BatchPrimer3”, with settings adjusted to give primers with Tm at least 65 deg, to include as much as possible (at least 800 bp) of each sequence in the amplicon.

The identified primer sequences were synthesized and placed as primer pairs in 96-well plate format. The primers were dissolved in water and prepared to give 5 uM each primer, while still in the 96-well plate format. PCR was in a 96-well plate in the same format as the primer pairs, using a master mix containing Phire polymerase, template consisting of a mixture of the P1 clones pVYS 174 C, E, H, I, and primers at 0.5 uM. (Note, Genomic DNA can be used as template, but locus specific clones such as BACs and PAC's are preferred). The products were analyzed by 96-well eGel, and show strong clean bands for 94 of the 95 wells. When Taq polymerase was used only 87 of the 95 reactions showed product.

Fragmentation of HER-2 Repeat-Free Sequence:

The contents of the PCR wells were combined and the DNA 1200mers were isolated by ethanol precipitation and PEG precipitation. The 1200mer mixture was sonicated by the same means used for fragmenting BAC DNA for other AM probes, and the sonicated product fractionated by HPLC with an ion exchange column to give narrow size fractions ranging from about 120 to about 400 bp. In some embodiments, the fractionation step is elminated

HER-2 Probe from 1-Step PCR Process:

To prepare Probe 5/13-76A the fraction centered at 150 bp was aminated by a standard procedure and labeled with Spectrum Orange.

HER-2 Probe from 2-Step PCR Process:

To prepare a HER-2 probe from the 2-step process, a portion of the same 150 bp HPLC fraction was treated with a blunting agent and ligated to adaptors. The adapted product was used as a template in a second PCR reaction, this time using only a single primer, with sequence corresponding to the adaptor. The use of a single primer to amplify the adapted template suppresses amplification of the shortest fragments via a stem-loop structure, and gives a product enriched in the longer fragments. The PCR product was aminated and labeled with Spectrum Orange, then subjected to 75° C. 72 h formamide treatment.

HER-2 PCR Probe Via Aminoallyl dUTP Amination:

To prepare a HER-2 PCR probe via aminoallyl dUTP amination, the HPLC fractions of sonicated 1200mer were recombined (to simulate unfractionated fragments), treated with blunting agent and ligated to adaptors containing common 5′ end containing a BspQI restriction site. The adapted product was used as template in a second PCR reaction, this time using two primers corresponding to the adaptor sequences, and with aminoallyl dUTP replacing half of the dTTP. The PCR product was treated with the restriction enzyme BspQI to digest off the common ends, and the DNA product labeled with Spectrum Orange, then subjected to 75° C. 72 h formamide treatment.

HER-2 Oligo Probe:

Design of the HER-2 oligo probe started with the same repeat-free sequence as the HER2 PCR probes. An application was used to select non-overlapping 80-base portions of this sequence, all with approximately 50% GC content (G+C min=39, max=41). A total of 401 such sequences were found. These were entered into the web application QuickFold with parameters Na+=0.05M, Mg++=0.001M, maximum of 1 Folding and the results copied as text and deltaG. The Find feature of Excel was used to flag sequences containing certain restriction sites for removal (to allow these sequences to be used in adaptors without interference with target sequence). The remaining sequences were sorted by deltaG, and the 288 (three 96-well plates) with lowest tendency to fold were selected for further processing and sorted by genomic location. The 80mer sequences were joined in pairs via a common 20 base connecting sequence and a different common 20 base was also added to the 3′ end of the pair, bringing the total to 200 bases to be synthesized. All 288 of the 80mers (Figure were represented in 144 separate “Forward” 200mers. The complements of the target 80mers and common 20mers were also calculated, and assembled the same way to give 144 separate “Reverse” 200mers. The assembly of these was designed such that upon mixing the sequences should duplex in a staggered fashion, giving long chains of annealed product.

Nucleic acids were chemically aminated by the procedure above, and labeled with Spectrum Orange. The labeled products were purified by gel electrophoresis to remove truncated synthesis products, and the full-length products combined to give the final probe.

Design of additional HER2 Oligo Probes started with the same sequence as for HER2 PCR probes, masking repeat sequences. Additional masking was introduced to flag for removal other undesirable subsequences: 5 or more consecutive “G” or “C” bases, and sequences corresponding to the BspQI restriction site “GCTCTTC” and “GAAGAGC”. The remaining sequence was processed to identify 60 base stretches with 55-65% GC. Very few acceptable sequences were found in the downstream 40 kb of the sequence, so only those in the range 1-180 kb were carried to the next stage. To the 3′ end of each sequence was added the bases “GGTTGAAGAG” polymerase using a complementary primer. The web based application “Zipfold” was used to determine the energy of folding. Of the 1027 60-mers assessed, the 960 with the lowest tendency to fold were retained. These were separated into 5 groups (192 members each=two 96-well plates) according to energy of folding, with each group sorted by position in the overall genomic sequence. The oligonucleotides were dissolved in water, and combined into groups according to folding tendency. Samples of each group were annealed with a primer containing sequence corresponding to the common 3′ end and Phire polymerase to elongate each to a blunt ended duplex mixture. These were purified by ion exchange HPLC to remove products of truncated oligomers.

p53 PCR Probes:

Same as with the HER-2 probes, the p53 PCR probes were designed based on the sequence of the BAC for the corresponding BAC p53 probe. Kilomers were generated by PCR, combined and fragmented by sonication. For the 1-step PCR probe the sonication fragments were simply aminated and labeled. For the 2-step PCR variants the sonication fragments were blunted and ligated to adaptors to make the template. To generate bulk DNA for amination and labeling the template was amplified using primers corresponding to the adaptor sequences. Bulk DNA containing the amines for labeling was prepared from the same template but including aminoallyl dUTP in the reaction mixture. For an additional variant, the adaptor sequences were removed by digesting the PCR product with a restriction enzyme specific to a recognition site included in the adaptors.

Generation of Repeat-Free Sequence in p53 Locus:

Sequence for the p53 PCR format was identified using the locus defined by the BAC clone pVYS 173i. This clone consists of 172 kb at hg18_dna range chr17:7435119-7606823. These coordinates were entered into UCSD Genome Browser, and the corresponding sequence presented using the Repeat Masker function to give the portions known to be repeat sequence in lower case. Table 1 below shows primer sequences.

TABLE 1

SEQ

ID

Direction
Name
Sequence
NO.

1
01
FORWARD
p53 AF01
TCCCCTCACGCTTCTCCTTCAGTTC
203

2
01
REVERSE
p53 AR01
AGGGTCAGGGATTGGGGAGCTAGTG
204

3
02
FORWARD
p53 AF02
TGATGAGGGGAAGGCTGTCTACCTGA
205

4
02
REVERSE
p53 AR02
GCTGACTCTCAGCCCCTCCTCCAG
206

5
03
FORWARD
p53 AF03
GTGAGGGGCGAGAAACAAGACAAGC
207

6
03
REVERSE
p53 AR03
TGCAGAAGGGAAGAAGGTTGTTACGC
208

7
04
FORWARD
p53 AF04
GCCCAGCCTTAACCCCAGAACTCAG
209

8
04
REVERSE
p53 AR04
GCATCGGAACTCTGCTCATGGAAAG
210

9
05
FORWARD
p53 AF05
ATGGCTCTGCTGACCCAACAAACAG
211

10
05
REVERSE
p53 AR05
AAAACAGGATGGCCTGGCTCAGTTC
212

11
06
FORWARD
p53 AF06
TCCCCATTTCTTGGAGTGGGATTGA
213

12
06
REVERSE
p53 AR06
TTATAAGCCACTCGGAAGCCCCTCA
214

13
07
FORWARD
p53 AF07
GGAGCCCTTAAGCAACTAGCCTCTCTCC
215

14
07
REVERSE
p53 AR07
GGGCTGGCCATAGCGAAAAACACTA
216

15
08
FORWARD
p53 AF08
AAAGTGTGAAAAGCGCCTGCCCATC
217

16
08
REVERSE
p53 AR08
CCTGGGGTTCAAAAACAGCCTGACT
218

17
09
FORWARD
p53 AF09
GGGCATCACTTTCTTTTCCCCCATC
219

18
09
REVERSE
p53 AR09
TTGAAGATCTGGCAGGCAGTGATCC
220

19
10
FORWARD
p53 AF10
CGGTCCTGCTCTGGTCAATAAAGGA
221

20
10
REVERSE
p53 AR10
GACCCTAGCCGGGCTGTCCCTAC
222

21
11
FORWARD
p53 AF11
GGGCCTTCACCTTGATAGGCACTCG
223

22
11
REVERSE
p53 AR11
AGCTAGCAAAGGGGGAGATTGCACA
224

23
12
FORWARD
p53 AF12
CAGTCACTTCGTCGCGGCTAAAACA
225

24
12
REVERSE
p53 AR12
GGAGCTGAGCTGTAGTCTCCGAGCA
226

25
13
FORWARD
p53 AF13
CAGAAAGGGTCACCCCCTTATGTCG
227

26
13
REVERSE
p53 AR13
GAACCAAGCATTTCATGGCTCACAA
228

27
14
FORWARD
p53 AF14
TTGTGGACTGTCCCTGACCTGGGTA
229

28
14
REVERSE
p53 AR14
CTCCTGCCATAGGACCCAAGCTACG
230

29
15
FORWARD
p53 AF15
ATGAGCACCTGCCTCTCTCTGCTCA
231

30
15
REVERSE
p53 AR15
CATGCATCTTCTCGGTGAGCCAGTC
232

31
16
FORWARD
p53 AF16
TGTTAAGCCGTGGATTCAAGGACCA
233

32
16
REVERSE
p53 AR16
GTTTTCCCTGTTGGTGGGAAGGTCA
234

33
17
FORWARD
p53 AF17
CCATCACCCAGGCAGTCATCTTCAT
235

34
17
REVERSE
p53 AR17
TGAGGAGAGCCTCTGGGATCTGGAG
236

35
18
FORWARD
p53 AF18
TGACCTTCCCACCAACAGGGAAAAC
237

36
18
REVERSE
p53 AR18
TTGCTTGGCAGTTGTGGCAAGTAGC
238

37
19
FORWARD
p53 AF19
AACCTCCAGATCCCAGAGGCTCTCC
239

38
19
REVERSE
p53 AR19
GGCTCTTGGTAGTCCTGTGGCTGGT
240

39
20
FORWARD
p53 AF20
CAGTCGGGAGAAGGGAGGGAGAGAA
241

40
20
REVERSE
p53 AR20
CACCACGTGCTGGGCAGAGACTT
242

41
21
FORWARD
p53 AF21
AGGCCAGCAGAAGAGCCCGATTAAA
243

42
21
REVERSE
p53 AR21
GCGCTGTTGGAATTGTAAAGCTTGC
244

43
22
FORWARD
p53 AF22
CCAGGTAGTCCAAGGTGCCCTTTCC
245

44
22
REVERSE
p53 AR22
CGGTGACTTCCCCACTGGCACTAAT
246

45
23
FORWARD
p53 AF23
GCGGAGAAAGGCAGGGGTGTAAATC
247

46
23
REVERSE
p53 AR23
ACACTCTGACTGTCCCTGGCCCCTA
248

47
24
FORWARD
p53 AF24
TGGAGGTGATCTTGAGAAGGGGTGA
249

48
24
REVERSE
p53 AR24
TCAGATGGGGAGAACCCTGAGGTTG
250

49
25
FORWARD
p53 AF25
GCCAGCTCTCAGCCATCACAGTCTT
251

50
25
REVERSE
p53 AR25
CCTCTAACCCTCATGGACGCAGACC
252

51
26
FORWARD
p53 AF26
GACTGAGTCTGGACGGCAGAGTGGA
253

52
26
REVERSE
p53 AR26
GCCCCTCTTCTGGAGCCTTGGTG
254

53
27
FORWARD
p53 AF27
ACGGCTGTTTTCCTCTTGGGGAGTC
255

54
27
REVERSE
p53 AR27
GCAAAGGCTCCGGTTTAAGCTCTGG
256

55
28
FORWARD
p53 AF28
CTTCTCGTCCTCGTCCAGCAGCTT
257

56
28
REVERSE
p53 AR28
CCCAAGCACCTGCTGGAATGACTC
258

57
29
FORWARD
p53 AF29
TGGCAAGTTTTAGCTTCAGTCGTCCA
259

58
29
REVERSE
p53 AR29
GACCTGCGCTGGAGCTGCTCTTATC
260

59
30
FORWARD
p53 AF30
AAAGAAGGCACAGGAGCCAGACAGC
261

60
30
REVERSE
p53 AR30
TCTTAATCTCCTGCCTTCCCCAGTGA
262

61
31
FORWARD
p53 AF31
GGGGTGCAGGTTGGAGGTTTTATGA
263

62
31
REVERSE
p53 AR31
GCTCAGGTACCAGACAGCTGGGTTC
264

63
32
FORWARD
p53 AF32
CACTGAACCCGAACCCCTGATTTTC
265

64
32
REVERSE
p53 AR32
ATTGATGAGCAGCTTCGGCAGATTG
266

65
33
FORWARD
p53 AF33
AGCTCGGTTGGGCTCCTCTCTCTTC
267

66
33
REVERSE
p53 AR33
GATTGTCCTCTGAGGGCTGGGATTG
268

67
34
FORWARD
p53 AF34
CCAAGCGAAGCTGCTCTACCTCCTG
269

68
34
REVERSE
p53 AR34
TCTTCCTTTAGACTCCCGAGGCTTGC
270

69
35
FORWARD
p53 AF35
ATGGAAGGCAAATCGCCTGAAACTG
271

70
35
REVERSE
p53 AR35
CGGAGGTAGGCCCTTCTCTCTCGAC
272

71
36
FORWARD
p53 AF36
AACCCCAGGCCCTGCTCCATAGTAG
273

72
36
REVERSE
p53 AR36
ATGCATGGCTTTGGCCTCCTTAGTG
274

73
37
FORWARD
p53 AF37
TGAAGTACAGGCAGAAACCACCCAAGA
275

74
37
REVERSE
p53 AR37
TCAGGCCATTATTGTCCCTGGCTTG
276

75
38
FORWARD
p53 AF38
CTGGGCAAAGGGGGAGGTGAAATAA
277

76
38
REVERSE
p53 AR38
GGCCCGCACTCAGACTGCTGCT
278

77
39
FORWARD
p53 AF39
CCCAACACCACATCAGGACATGTAA
279

78
39
REVERSE
p53 AR39
GCCGAATTCGAAAAACTCTCGGATCA
280

79
40
FORWARD
p53 AF40
CCCCCTCCATATACCCTTGCTTCTTCA
281

80
40
REVERSE
p53 AR40
CCGGAATCCCAGCTCCACTTACCAG
282

81
41
FORWARD
p53 AF41
CTTCTGGAAGCCTGTGGGGAGACCT
283

82
41
REVERSE
p53 AR41
ATGCAAATGCCCCCAAGAGGTAACA
284

83
42
FORWARD
p53 AF42
CACATAAGGAGGTGAGTTCCGACGTG
285

84
42
REVERSE
p53 AR42
AACTGCGATGAAAAGGGGTGCTGTC
286

85
43
FORWARD
p53 AF43
GCGGGCCCAACCTCTCCATATTTAC
287

86
43
REVERSE
p53 AR43
AGGCCCCGAGAGGGAGTGTGAG
288

87
44
FORWARD
p53 AF44
CTTTTGGGTGTGTGGAGGGCTTCAG
289

88
44
REVERSE
p53 AR44
ATCCCAGGGGCTGGAGTTTGAGTTC
290

89
45
FORWARD
p53 AF45
GGTTCTTCACGGCGGAAGTTGTCTG
291

90
45
REVERSE
p53 AR45
TCCTGGTCCTAGGGCACAGTGAAGC
292

91
46
FORWARD
p53 AF46
TCTGAGATGGAAGGATTGGGAGTCCA
293

92
46
REVERSE
p53 AR46
TGAGGGACTGCACACAGAGGGAAGA
294

93
47
FORWARD
p53 AF47
GGCTCTGTGATCAGTCCCAGTGCAG
295

94
47
REVERSE
p53 AR47
GCATCCAGCCTTCTAACTGCAGAGC
296

95
48
FORWARD
p53 AF48
TGGGAAGAAACTGCGGAATGAAACA
297

96
48
REVERSE
p53 AR48
AGGAGTGGAGCTTTGGGGAACCTTG
298

97
49
FORWARD
p53 AF49
AGTGAGCTGCTCCGGCAAAAAGAAA
299

98
49
REVERSE
p53 AR49
GCAAAAGCTTTCTTCCTTCCACCCTTC
300

99
50
FORWARD
p53 AF50
GGGAGACAGGTCTGAAGCCTGGAGAA
301

100
50
REVERSE
p53 AR50
TTGGGTGCTGAACTCTGACCAGGAA
302

101
51
FORWARD
p53 AF51
AAAAATGCGGACTCTGAACTGATGC
303

102
51
REVERSE
p53 AR51
TGCTGCCACAAGAAATTCACTACTTTTT
304

103
52
FORWARD
p53 AF52
GTGCGCGAGGTGAGGGAGGTGT
305

104
52
REVERSE
p53 AR52
GCTGTGGGGAAGAGTTGGGAGACAG
306

105
53
FORWARD
p53 AF53
CGGGCACAGCAGGAAGCAGGTC
307

106
53
REVERSE
p53 AR53
GAAGTGGGATCCGCATGTAGGCAAG
308

107
54
FORWARD
p53 AF54
CCAGCCTCCCTCACACTTCTCTGCT
309

108
54
REVERSE
p53 AR54
CCTGGAACCTATGGGGAGGAGGGTA
310

109
55
FORWARD
p53 AF55
ACTGGTCTGGACCACCCTCCACACA
311

110
55
REVERSE
p53 AR55
GAAGACCCAGAGAGGGGCTGAGACA
312

111
56
FORWARD
p53 AF56
TGCCCCAAACCTCCTTCTCACTTGT
313

112
56
REVERSE
p53 AR56
CTGCCACGCCCAGCAAGAGCAG
314

113
57
FORWARD
p53 AF57
ACCCTCCCTCCCTCTTCAGTTTTGG
315

114
57
REVERSE
p53 AR57
GCCAACCAAAAGGTGGGCTGTTC
316

115
58
FORWARD
p53 AF58
CCCCTTCTGCCCCCACTATGAGAA
317

116
58
REVERSE
p53 AR58
TGAAAGGAAGTTCTTCCCGCCCTTC
318

117
59
FORWARD
p53 AF59
AACCACTCCCCTCAGTCTGCCAAAA
319

118
59
REVERSE
p53 AR59
CCGCCACCATGGCAATATCAACTTC
320

119
60
FORWARD
p53 AF60
TGGACTTGAATTGGGACAAAGGCTTG
321

120
60
REVERSE
p53 AR60
TTCCCTCTTGTCTCTCCACCTGTGC
322

121
61
FORWARD
p53 AF61
GAAGGGATGTGGCTGATCAGAAGGA
323

122
61
REVERSE
p53 AR61
TCCTGTGCCTGTAGTCGTTTTGCTG
324

123
62
FORWARD
p53 AF62
AGTGGGAATTTCTGATGCGGAATGG
325

124
62
REVERSE
p53 AR62
GGGAAAGGGAGCCAGAGGCTAAGGT
326

125
63
FORWARD
p53 AF63
GTGGCTGCCTCCTCCCTCATCAAT
327

126
63
REVERSE
p53 AR63
TGCTCAATCCGATTAAACGCTGCTG
328

127
64
FORWARD
p53 AF64
CCAGCTCACCCCAAATCTGCTGTTC
329

128
64
REVERSE
p53 AR64
TGGCAGTCCCTGGTACTCCTGAAGA
330

129
65
FORWARD
p53 AF65
TGCAACCCTGGCTGTTTCTCTAGCC
331

130
65
REVERSE
p53 AR65
GGGAAGACGGGAACTGGAGTTTTGG
332

131
66
FORWARD
p53 AF66
ATTTTTAGCCCAGTGCCCCGAAGAC
333

132
66
REVERSE
p53 AR66
GTGCTCTCAGGGAGATCCCAGCAAT
334

133
67
FORWARD
p53 AF67
CAAGGAGGAGCCTGGTGGGTACTTG
335

134
67
REVERSE
p53 AR67
GGAGCTGGGAGAGGCAGAAATCCTT
336

1
01
FORWARD
p53 BF01
GCCATGGAAACGTCACAGTTCATCC
337

2
01
REVERSE
p53 BR01
CTGCGTGGGGGAAGGACACATTGTA
338

3
02
FORWARD
p53 BF02
TCCACCTCCAAGCCCAGATTCAGAT
339

4
02
REVERSE
p53 BR02
ACTTTGCCCAAAGACCCCTGTGTG
340

5
03
FORWARD
p53 BF03
ATTCCAACCAGCCTCTTCCGCTGAC
341

6
03
REVERSE
p53 BR03
AGACGGTGAAGGTGGGGACCAATTT
342

7
04
FORWARD
p53 BF04
TTCTCCTCCACCATGTTAGGCTTGG
343

8
04
REVERSE
p53 BR04
CAGGGCGCAATCTTCCTACTCCAAA
344

9
05
FORWARD
p53 BF05
CAACAGGGCCCAATTCTGAGAGAGG
345

10
05
REVERSE
p53 BR05
GGGACTTTTTCTTCTAGCCTGCCTCCA
346

11
06
FORWARD
p53 BF06
GGCAGAAAATGCTAACCTGCCCAGA
347

12
06
REVERSE
p53 BR06
CCCATTTTTGCCTTACTTCCCTCATCA
348

13
07
FORWARD
p53 BF07
GGCCTCTGATTTTGCTTCCCACCTT
349

14
07
REVERSE
p53 BR07
CCTGCTTGAGTCCCAGGTCCAAAGA
350

15
08
FORWARD
p53 BF08
CAGGGGTGGAACCCTAGCCAAGACT
351

16
08
REVERSE
p53 BR08
GTTCCCTGGGCTGGAAACCTTCCTA
352

17
09
FORWARD
p53 BF09
ACAGAGGCAAGGACAGGGACTGAGC
353

18
09
REVERSE
p53 BR09
TGGCCATCTGCTCCATCAGAAAGTG
354

19
10
FORWARD
p53 BF10
AAGGGGCTCCTGCTTGCACCTTC
355

20
10
REVERSE
p53 BR10
GGAACAAAGGCTGGAGACTGGGTCA
356

21
11
FORWARD
p53 BF11
TGAGCTCCTCAACCCGACTCTCCTC
357

22
11
REVERSE
p53 BR11
CATCTGCATTTTCACCCCACCCTTC
358

23
12
FORWARD
p53 BF12
TCAGGCAAAGTCATAGAACCATTTTCA
359

24
12
REVERSE
p53 BR12
TTAACCTGTGGCTTCTCCTCCACCT
360

25
13
FORWARD
p53 BF13
GGTTTCTTCTTTGGCTGGGGAGAGG
361

26
13
REVERSE
p53 BR13
TGGCCTCATCTTGGGCCTGTGTTAT
362

27
14
FORWARD
p53 BF14
ACCCTGTCAGCTGTGGAGCTTTTGG
363

28
14
REVERSE
p53 BR14
AGATCCCAGCACAGGAGCTCAGCAT
364

29
15
FORWARD
p53 BF15
CAGAGCGGCATCATCTCCTGCATAG
365

30
15
REVERSE
p53 BR15
TCCTTCTGGGGCAGAAAACTCAACA
366

31
16
FORWARD
p53 BF16
CTGCACCCTAGCCTGCCTCTCCTG
367

32
16
REVERSE
p53 BR16
AGGAGAGTGCTCCTGCTCCCCTCAG
368

33
17
FORWARD
p53 BF17
GCATATGAGTGAGGTGGGGGACCAG
369

34
17
REVERSE
p53 BR17
TCGTCCCTTCCCTTTGATTGTGAGG
370

35
18
FORWARD
p53 BF18
TGAGCAGGAGCTGGGTCAGACTGTT
371

36
18
REVERSE
p53 BR18
CCAAGTGCTGTTGTTACCTGGGGGTTA
372

1
01
FORWARD
p53 CF01
AGGGATGGGCCCTGAGACCTGTT
373

2
01
REVERSE
p53 CR01
GCCCAGTGATTGTGCAGTTGGATCT
374

3
02
FORWARD
p53 CF02
TGTCTGTCTCTGACATGTCCCTACTCAGC
375

4
02
REVERSE
p53 CR02
GTTCCCTTTCCTCAGACCAGCTCCA
376

5
03
FORWARD
p53 CF03
GGATGGCACTCAGGTGGGTGGTAG
377

6
03
REVERSE
p53 CR03
CCCAGAGGTTTCCTTCCCCTCAAAA
378

7
04
FORWARD
p53 CF04
GCCAGTAGGTGGAGGCATAGCGAAG
379

8
04
REVERSE
p53 CR04
GAGGGAGGTAGTGCAAAGGTGGCATT
380

9
05
FORWARD
p53 CF05
CTGGTCTTGGGACTCCCTCTTCAGC
381

10
05
REVERSE
p53 CR05
GGTGGAGCTTTAATGGGAAGCGTCA
382

11
06
FORWARD
p53 CF06
CAGCAAAAACCCCGACAGACAGACA
383

12
06
REVERSE
p53 CR06
GGTAGGGGCTATGGGCTTGGATACG
384

13
07
FORWARD
p53 CF07
TGAGACCAGTTTCCTGCCTCTGTGG
385

14
07
REVERSE
p53 CR07
GGCTTTAGCCCCAGGGCTCCTTAGT
386

15
08
FORWARD
p53 CF08
GGTCTGGATGCCCATCTTCGACAAC
387

16
08
REVERSE
p53 CR08
GAACAGGGTCCCAAGGACAACGAAC
388

17
09
FORWARD
p53 CF09
TTCTTTGCTCCTAGAGGCCCCATCA
389

18
09
REVERSE
p53 CR09
GATGAGGGGATGGTCTGTCCCTGTC
390

19
10
FORWARD
p53 CF10
TCCAGGGAGGCAGACTTGAGACAGG
391

20
10
REVERSE
p53 CR10
CAAAAATACCTTTGGGGTGGGTGAGG
392

21
11
FORWARD
p53 CF11
GTGCGGGTGGTGAGCTAGGGAAG
393

22
11
REVERSE
p53 CR11
ACTCTCAGGGGATATGCAGGCAGGA
394

The upper case “unique” sequence portions were copied as above to select out the upper case “unique” subsequences, retaining the position information of each. This yielded only 17 sequences longer than 1200 bp totaling 35930 bp. To these were added 13 fragments with lengths 800-1200 and 10 fragments 650-800 bp to give a final total of 55661 bp. The application “SeqChop3” was used on the first set to generate 1200-mers with 400 base overlap, yielding 65 sequences. The second set (800-1200) for 800mers with 200 base overlap gives 26 sequences. To complete a 96 well plate an additional 5 sequences in the 650-800 base region are accepted. The sequences so generated were adjusted to give primers with Tm at least 65 deg, to include as much as possible of each sequence in the amplicon. The primers were dissolved in water to give 5 uM each primer, while still in the 96-well plate format. PCR was in a 96-well plate in the same format as the primer pairs, using a master mix containing Phire polymerase, template consisting of the BAC clone pVYS 173i and primers at 0.5 uM. At the conclusion of the PCR, additional primer, Taq polymerase and dNTP was added and the plate subjected to an additional 8 cycles to increase the product yield. The products were analyzed by 96-well eGel, and show strong clean bands for 91 of the 96 wells. The contents of the PCR wells were combined, and the DNA isolated by ethanol precipitation and PEG precipitation. The mixture was sonicated by the same means used for fragmenting BAC DNA for other probes.

p53 probe from 1-step PCR process:

To prepare p53 probes from the 1-step PCR process, the sonicated product was aminated and labeled with Spectrum Orange, then subjected to 75° C. 72 h formamide treatment.

p53 Probe from 2-Step PCR Process:

To prepare p53 probes from the 2-step PCR process, a portion of the sonicated product was fractionated using disposable silica based spin columns (PureLink PCR Purification Kit) but adjusting the binding buffer to isolate a fraction ranging from 100-300 bp. A portion of this fraction was treated with a blunting agent and ligated to adaptors. This adapted product was used as template in a second PCR reaction, this time using only a single primer, with sequence corresponding to the adaptor. The PCR product was aminated, labeled with Spectrum Orange, and subjected to 75° C. 72 h formamide treatment.

p53 PCR Probe Via Aminoallyl dUTP Amination:

This probe was prepared as described above except the PCR step included aminoallyl dUTP added to the PCR mixture. The PCR product was labeled with Spectrum Orange and subjected to 75° C. 72 h formamide treatment.

A portion of the sonicated blunted repeat-free p53 DNA was ligated to adaptors containing a BspQI restriction site. The product was used as template with a pair of primers corresponding to adaptors in a PCR reaction containing aminoallyl dUTP and the product exposed to BspQI restriction enzyme to remove the adaptor ends. The digestion product, containing only the sequence specific to p53, was labeled with Spectrum Orange and then subjected to the 7° C. 72 h formamide treatment.

Oligo-PCR Probe for p53

Sequences for p53 Oligo Probe were identified as above; however in this case, in addition to masking repeat sequence, all subsequences of 5 or more consecutive “G” and “C” were masked. Parameters were set to identify subsequences of length 140, with GC content of 45-60%. The 289 sequences identified were subjected to NCBI BLAST to find matches to other loci in the human genome. 281 unique sequences remained after this. To 280 of these was appended a common Forward and Reverse sequence, each 25 bases, and containing restriction sites, to give 280 sequences each 190 bases. These sequences were sent to IDT for synthesis as “Ultramers” in 96-well plate format. The products were combined and used as template in PCR with primer sequences corresponding to the adaptors. The adaptor ends were removed by digestion with the restriction enzyme, and the product aminated, labeled with Spectrum Orange, and subjected to 75° C. 72 h formamide treatment.

Table 2 shows the 190 by sequences.

TABLE 2

Sequence

Name
Sequence
SEQ ID NO.:

p53F193
TAT ACG CAG TGC TAC CAT CGA TCA CTT GCA GTG GGA ATT TCT GAT GCG GAA TGG
395

TTG ACA TCA TAT CTG GAA TTT TAA TTA GAA TGA AAA ATG GCC CTT CCT AAG GGG

CTA TAT GGG CCA CCG TGG ACA TGG GGC TTG GGA GAG TTT TGA GAC CTG GGA

GAA ATG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F194
TAT ACG CAG TGC TAC CAT CGA TCA CAA GGA AAA GGG ACA CAG AAT GAA TGG GAA
396

TGA CAA CTG GGC AGC CAG AGA GCT ACT TGA AAG GTA GCA GGG AAA GTG GAG

TTC TGA AGG GAG TTC TGA GGG GAC CAG GCC CCA GGT ACC CCA ATT CCC ACA

GGG AAT CGA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F195
TAT ACG CAG TGC TAC CAT CGA TCA CAG AAG GAT GAG TCA GGC TGT AGA GTA ATC
397

TAC TCT GGT TCT CAC GGG ACC AGC CCA GGG CAC AGC CGG CAG GGA GGC TGC

TGG CAT AGA GGC CCT GTC TCC CCT GGC CTC TGT CTC TGA TGA ATG ATC CGG ACA

GCA GGC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F196
TAT ACG CAG TGC TAC CAT CGA TCA CCT CTC CGT GGA GAT GGC TCT GAG AAA TCA
398

AAT ATT GAC AAT GAG GGA ACA GAA CTT ATT AAA TCT GGG ACA GGG ATG TGT GTG

GGG AGC TGT GGG AAT GGC CGG ATG CCT GGG TTC GGA GGG TAA TGA AGG TTC

TGG GAG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F197
TAT ACG CAG TGC TAC CAT CGA TCA CGT TTA AGA AAA GAA ATG GGG TCT GTG CAA
399

AGT TCC TTT CCC CGA GGG ACC CAT GAA CCT TGC CCC TGA GGC TCC CCA CCC

CAC GCC TCC AGC AAA TTT TCA CTC CTT TGG GAC TTT TCC CTA GGG GTT CTG GCC

AAC CAC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F198
TAT ACG CAG TGC TAC CAT CGA TCA CTC ACC TCT GTC TGC CAC CCA TGG CCT ATC
400

TGG CTA GAG GAA CAC CTC CAC GTT CCA TCT TAG CTG TGC TAG CTG TGC AGC TGG

TTT TGG GGT GGG CGG TAC TCC TCT CCT TCC ATT CCT GCT GTC AGC AAC AGG AGT

CTG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F199
TAT ACG CAG TGC TAC CAT CGA TCA CGA GGG AAA GGG TTG ACA CTT CTG GGA ATA
401

AAG ATA CGG TAG GTG GGA GGG TGA GGT CAG CGC TGG GAT AGG GTG TGG TTA

ATC CAC TTG CTC TTC AGG AGT ACC AGG GAC TGC CAC TCC TGG TAG TGC CAG GCT

GGC ATA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F200
TAT ACG CAG TGC TAC CAT CGA TCA CAA CTG AGG TGC CAG CTT CTC TTC TCC TCA
402

TCC AGC AAA GAA AAT GTC ATA AAT TTC GCT CCT CAT GGA AAT GTA ATC AGT GGG

CTC GCT GCT GGC TTT GTC TTA ATT AGG CAC TAT TGA TGG AAA CAG GGA GGG CGC

CTT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F201
TAT ACG CAG TGC TAC CAT CGA TCA CGG AAG AGC CAT TCT GAG GGG AAA TTT GCC
403

TGC TGG TAA CCA GTT TAA GGA TAC CAG CTG CTG GTA TAA ATA CTG CTG GAT AAA

TAC TGC TGG ATT TAT ACT GCA GGA TAA ATA CTG CTG GCC CCT GCG GTA TTT CCT

AGT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F202
TAT ACG CAG TGC TAC CAT CGA TCA CCT AGA GTG AGC CCC GAC TTA GCA GAG CAG
404

TTC CTC CTG GGG CCT GCG GTG TGG GAT CGC GTG GTG AAC CCC ACG GTG CAT

GCG CCT CAG GCT CTA GTT TGA GGC AGG AAA GCG CAG CTT GAT GCT TCT CTG

GAG ACT GAT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F203
TAT ACG CAG TGC TAC CAT CGA TCA CGC TGT AGT TGG TTT TAT TGA TTT GCT GGC
405

CTA ACA GAA CGT TTT TCC TTG GAG CAA AGT ACA AAT CCT TCA AGT TTG AAA TTC

ATA ACC TGA GAT CAA TGC CTG TGG CAG CCT GTG GGG ATG AGG AAG GAG AGC

CAC AGG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F204
TAT ACG CAG TGC TAC CAT CGA TCA CGC CGT TAG GCT GCA GCC TAA TGA AAA GAG
406

AGT GCC CAG CGC CTC AGA CTT TGC GCC TGG GAT TCT GAG CAC CTG TCC GAG

ATC CCC GCT TCC TGC CAT CCT ACC TTT CTG AGA GAG GCA CCA CTG TGA CCT TCC

TCG TGC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F205
TAT ACG CAG TGC TAC CAT CGA TCA CCC AAT CTG TTC TCA GGG CAT TTT GAG TCA
407

AAT AAA TGA TCC TGA CTG ATC TTA ACC ATT AGC ACA GAG TTC CTC AGC CAA CTC

TGC TAA GAG ACC TCA GTA CAC ACA AAA CAG TGT TCC TGC CCC TCA GGA CTT CAA

AGC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F206
TAT ACG CAG TGC TAC CAT CGA TCA CAT GTG CAC CCC TTC CTT GAT CCT CTC TCA
408

CTC ACT CAT GGT CCT GGA GGG TAG AGT GGA TGA GGG TTT GGG CAA CCA CAC

TTC AGC TTG ATA GAT CTT TTC CTG ATT ATC TTA TGT TCT CAT ACC CCG GGG CAG

AGA TAG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F207
TAT ACG CAG TGC TAC CAT CGA TCA CTG GCC ATC ATC TTT AAC TCC CAT TTG TTC
409

CTA GTG CCT TGA GGG ACT GAC TTT GAT TTT TAG CCC AGT GCC CCG AAG ACA GTA

GGA TAT CCT AGG GAA AAA CAG CAA GGC CTC CCA GGC CCC TGT GGA GTA CAG

AGC CCT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F208
TAT ACG CAG TGC TAC CAT CGA TCA CCT GGT TCA GGG GTG GTA TCT GCT TGT AGA
410

CTC TCC ACT CCT GTA TAC CTG TAG GTT TTG CCT GCA CGA TGT CCA GCA AAG CTG

AGA AGA AGC AGC GAT TGA GTG GCC GAG GAA GCT CCC AGG CAA GCT GGT CAG

GGC GGG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F209
TAT ACG CAG TGC TAC CAT CGA TCA CTC CCC AAG GAG GAG CCT GGT GGG TAC TTG
411

CTG GGG CAG AGG ATG CTT AGC AAT GGA GGG TGG GGA AAG TCA GAG GGG CTT

GGA GGC ATT TTA GGG CTG GGG AGC AGG CGC TGT TGC TTC TGG GCA GGA AAC

GGG CTA AGT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F210
TAT ACG CAG TGC TAC CAT CGA TCA CAG CCA TGG AGG TTC AGC AGC CCT GCC ACT
412

GAG TCC TTT TTT TGT ATG TCT TCC TCC TGG CCA GCC TCA TCC TTG TTC TTT CTG

TCT TAA AAT TTC CCA CAA TAT ACC AGG AGT TCA CCA ACC AGT CCT TTC TCT AGC

CTC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F211
TAT ACG CAG TGC TAC CAT CGA TCA CTC CTC CTT ATT TCA AGT GTT CCT GGT TGC
413

TCC TAC CCC ACT GAA GAG GGT TTG ATC TCT TCC TTT TTC CCA TTT TCA CCC TGG

GTG GCA AAA CAA ATA ATG TTT TTC TCC CAT TAA GCC CAT CAC CAT GGA GAC TCA

GCC GAC TTC GAA AGA CGC TCC ACG ATA T

TAT ACG CAG TGC TAC CAT CGA TCA CGT TTT GCC ACC CTT TGA ATC CAA AGC CCG
414

p53F212
GCT GAT GCT TTC TAC CAT CTG TTG AGG GCT GTT CTA TCT CCG CCC TCA TTT TGG

CAG GGA CTT GGT AGA CTG CGG AGG TTC AGG TTC AGG ATG ACA GGA AAG AAG

TCT AGA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F213
TAT ACG CAG TGC TAC CAT CGA TCA CCT AGG GAG GAA AGA GTT TAA CGA GGC AGC
415

CTC TTG TGG CTT TGA CCT GAG GGA TTC CTT TCC TGT GGG TGA GCC GGA GCC AGT

AAG GTT GGA AGG TTT TTG GTA GTT GTT GCT ATT GCT GGG ATC TCC CTG AGA GCA

CTG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F214
TAT ACG CAG TGC TAC CAT CGA TCA CAG ACA GGA CTA TGA GCT AAG TGT AGA CCT
416

CCT TCT TTA CCT GGA GCT CCT TTA CAC TGA TAG TAG CTT GTG AAA TAG AAT CCC

CTT CTA AAA TTA AAG GTT GAG AGG TTA AGC GTG GGC ATG GGA GAA AAG TCC CTG

GGC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F215
TAT ACG CAG TGC TAC CAT CGA TCA CAT AAG AAC TTG CAT TAG GTT CCA GGA TCT
417

TAG AAA TGT GCA GAA TGT ATC CTT CTG AGT TCT GAA GTG CTC AGG TGA AAG CAG

TTA AAA TGG GAT TGC TCG CTG CTA GGA GGA GTA GGG GAA GAT GGG AGG GAA

GAC AGT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F216
TAT ACG CAG TGC TAC CAT CGA TCA CTC CCC TGG CCT AGT TCT TGC CTG GAA CCT
418

GAA TGC CAG CAG TTT TCC CAA GGG AGT TGG ATC ATC TCA CCT ACT CAC CTA CTA

CTA AGC TCC GGA ATC GCC TGT TCC ATC TTT TCA CGC CTT CCT GCC TCG GCC TCT

GTG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F217
TAT ACG CAG TGC TAC CAT CGA TCA CCT CAT TCC CCT CTT AGT TTT CCT TTC TGG
419

AAG CCA GAG GGA GTT TCC TGT TCC TCA TGG TCT TTT GGT TAT ATC TCA TTT GTT

CCT TCC TCC TTT CAG CCC CTG GGG AAT GGG AAA GCC ACC AGT TTT ACT CCA ATC

TCC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F218
TAT ACG CAG TGC TAC CAT CGA TCA CGT GTG ACT AGT AGA TCA GGA GGC AGA GAA
420

ACA GAG CCG CTG GGT TTT ATG GGA GTC AGA GTG GGT GTG GCA GAA CTG AAT

GCT CAG CAG GCC AGT GGT TTG GAG AAG GGC TGA TTC CTA TGA GCC CCA ACT TCT

CCA TGT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F219
TAT ACG CAG TGC TAC CAT CGA TCA CGA CTG TGT TTA ACT TTC CAA GAA ATG CCT
421

TCC TTT TTG TAT GTT TAT TCT TCC TAG AGC CAC GGT TGG AGG GAC CTC AAG CAC

AGA GTG AAG AAT CAG TGG AGC CCG AGG CAG ATG TGG TAG GCT TGG GTC TTC

CCT GTG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F220
TAT ACG CAG TGC TAC CAT CGA TCA CCT GGT GAA TCA AAG GAT CAG TTG TGG ATG
422

GCA CAT GAG GAA TTC TCT CTT GTC TGC CAC TTC TTC TAC TTG CTC TCT GCT GTT

AGC CTA CTC ACC ACA AAT CCA GTT CAC TGA GTA AAA GAC AAA GTC ATC TGT GCC

TTC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F221
TAT ACG CAG TGC TAC CAT CGA TCA CCC AGG ACA GAG GAG GGC TTT AAG TAA GGC
423

GCA GGT CTC TCT TGT CTC CCC ACC TTG GGT CAA ACT GTG ATG AGC TTA TTC ATC

ATG CGG GTG AAC ACC TGA ACA AGT TGA ATG AGC TGG GAA GAT ATT GTC CTA ATT

GGA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F222
TAT ACG CAG TGC TAC CAT CGA TCA CTC TTT TGG ACT CCA TCG TGA AGA AAT TGC
424

TCC TAT ACC CTC TCC CTT TTC CAG CTT GTG GTC TCC CTG CTC CTA ATT GCT TGA

TCT AGG CTA ATA TCC TAC ACA TTC CCT TAA TTC ACC TTT ACC TTT GAG ATC GAG

GGC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F223
TAT ACG CAG TGC TAC CAT CGA TCA CCT GTT TCT TCC AAA GCT GGA AAG CTA TCT
425

TTC AGT TTC TCC TAA CCA TTC TGA ACC TGT CTG GCT TCT CTC ATC TCC CAT GTC

CTC ACT TGT GAT TGT GGA TGG GAG GAA TGA CTC ATC CTG GCA GTT GTA GTT ACT

TTG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F224
TAT ACG CAG TGC TAC CAT CGA TCA CCC CTC TGC TCT CCT TCT GTA GAA GCC CCT
426

CTT CCT TTC CCG AGC TGC GCT GAC AGG ACT GGC GGA TGC AGT GTG GAC ACA

GGA GCA TGA TGC CAT TCT GGA ACA CTT TGC CCA GGA CCC TAC AGA ATC CAT CCT

CAC CAT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F225
TAT ACG CAG TGC TAC CAT CGA TCA CTT CAT TGA CCC TTG TTT TGG GCT GAA GCT
427

AGA GCT GGG CAT GCC TGT ACA GGT GCG TAC CCT ACA TTC CCA GAT CAA AGG

TGG TCC TAC AGA GAG CCG AGG GCT CTC AGT TCC TTC ATC ATC CTT AAG TCT TGT

CAT CTC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F226
TAT ACG CAG TGC TAC CAT CGA TCA CCA TAC TCT CTG CAT TTC CTC CCC ACA TCT
428

CTG GAT ATG GAA TCA TAT CCG TGA AAA GAA CAG GAA GCA CTT CAT GGC ACT TCC

ATT TAG AGA AGG ATC AGG ATA CCC TGA GGG ACG TGA GAA AGA ACT CAG CCG

GGC TTT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F227
TAT ACG CAG TGC TAC CAT CGA TCA CGT GAC TGG GTG TCA TGG CTG GGC CTG
429

GAG ACT GAA CTC TGA TCT GCT GTC TTC CAT GCA GAC CCA GAA CCA GCT TGT CTA

CTT CAT TCG CCA AGC ACC AGT TCC CAT CAC CTG GGA GAA CTT CGA GGC AAC TGT

GCA GTT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F228
TAT ACG CAG TGC TAC CAT CGA TCA CGG GAC GGT GCG GGG CCC CTA TAT CCC
430

GGC CCT GCT TCG GCT GCT CGG TGG AGT CTT TGC CCC TCA GAT CTT TGC AAA CAC

AGG CTG GCC TGA GAG CAT TAG AAA TCA TTT TGC TTC TCA TCT GCA CAA GTT CTT

GGC CTG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F229
TAT ACG CAG TGC TAC CAT CGA TCA CAA AGG GAA ATA TAG GTG CTG TGG GGA CAC
431

TGG GAG GAC TCA CAT GTC TCG TGG TAG AGA GTG TTG AAG GCG CGA TAC AGA

TCT TTG ACC TTA TTT TAT GGC AAT TTC TTG TTA TAA CTC CAT GAG GTT TCC CTC

TTG CCT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F230
TAT ACG CAG TGC TAC CAT CGA TCA CTG GTC ATT TCT TGA TAT CAA AAA AGC ATG
432

AGT CAC CAT TTC TTC TAC CTG CTC TCC TGA AAT GCA TAT GCT TCC TGG TCC CCT

ACC GAT CAT CAA CCG TGA TTC CTA CGG GGC CAG CAA GAA CTC ATC CCC TCT GTC

CAC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F231
TAT ACG CAG TGC TAC CAT CGA TCA CTC CAC AAA CTT TCC TTT CCT TAG ACA CTC
433

GGT ACA AAC TGG AGG GGC ACA CGG TCC TCT ACA TCC CTG CAG AGG CCA TGA

ACA TGA AGC CTG AGA TGG TGA TAA AGG ACA AAG AGC TGG TGC AAC GGC TAG

AGA GTG AGT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F232
TAT ACG CAG TGC TAC CAT CGA TCA CGC TGG CAC TGC TAG CAT CAC CTG GCG ATC
434

ACA GGG GAG AGG GAA AGG GGA GGC TCG GAT GCT GAC AAT GGA GTT GGG TTA

GGA GGT CTC TGT CGG GGT TGC GGG GAG TGA AGG ATG CTG TTG GGA AGC AGT

GTG AAG AAA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F233
TAT ACG CAG TGC TAC CAT CGA TCA CGA AGG AGA TTT TGT ACT CTC CCC TGC AGC
435

CTC CAT GAT CCA CTG GAC CCG GCA GAT AAA GGA GAT GCT CAG TGC CCA GGA

GAC TGT GGA GAC AGG AGA AAA TTT AGG TCC TCT GGA GGA GAT TGA GTT CTG

GCG CAA CCG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F234
TAT ACG CAG TGC TAC CAT CGA TCA CTG CAT GGA CCT GTC TGG CAT CAG TAA GCA
436

GCT GGT GAA GAA GGG AGT GAA GCA CGT TGA ATC CAT CCT GCA CCT TGC CAA

GTC GTC CTA CTT GGC GCC CTT TAT GAA ACT GGC ACA GCA GAT CCA GGT TTG TGA

GCG AAT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F235
TAT ACG CAG TGC TAC CAT CGA TCA CAA AGG ATT CAG GCT CAG CAA GAA GTG GGC
437

AAT GGT TGG GAT GAT ACA GGG AGC TAA GTA AGG AGA GGG AGC CAA GGC AAT

CTT CGA TAG CAC AGA CTG ACC CAC CCA GGG TTC GGC CTT GTA CTT GCA GGA

GAG TAT AAA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F236
TAT ACG CAG TGC TAC CAT CGA TCA CGT AGA ATA GAG GGT CCC TAA GGA GAG AGA
438

CAT CAA GTT AAC CAA TAA ATA TAC ATC AAG TAC CTT CTG TGA GCA CAA CCC CTG

CCA CCC AGC TGG GAA AAT AAG ATT ACC CAA AAC AGG ATT ACT AGA CAG CGT GGG

AGC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F237
TAT ACG CAG TGC TAC CAT CGA TCA CAT GAT GTG ACA GGC TGA TTA TGT TAG TGC
439

TAG GTC ACA CAG GAG GTA AGA GTG GCC TGT AGA ATA CAG ATA GAA GAC CTG TCT

TCC TAG GTG AAA ATT TCA GCT GGG CAT TAA GGG AAT TGA GCC AGA ACT AAC TGG

AGG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F238
TAT ACG CAG TGC TAC CAT CGA TCA CGC CTA AAG CCC TGG CTG AGA ACA GGG
440

CAG TGA AAG GGA ACT GGG TGA CAA CTA TGG GGA CGA ATG AGA GTG ATA TGC

GGC CAG TTG ATC CGA CAG CAA CAG AGT ATC CAC TTA GTG CGG AGT CCT GAA TTC

AAC TGG AGA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F239
TAT ACG CAG TGC TAC CAT CGA TCA CCA GAC ACA AAA TCT AGA AAA ATC TAG ACT
441

CGA GGG AAT TCT TAG TCT GGT AGG GGA GCT AGG GCA CAC ACA TGA GAA AAG

AAA AGT TGA CAT GTG CTA ACT CTG GTC CGA AGG CTC ACA GAG ACC TGA AAT GAT

CGC TAC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F240
TAT ACG CAG TGC TAC CAT CGA TCA CTC AGT GCT TTC TAG TGC ATA AAT CTA TTT
442

CTC ATC TCT CTT TAC TGC CAG GAT GGC TCT CGT CAA GCA CAG TCA AAC CTG ACC

TTT TTG TCA ATC CTG AAG GAA CCT TAC CAG GAG TTG GCT TTC ATG AAG CCC AAG

GAC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F241
TAT ACG CAG TGC TAC CAT CGA TCA CTC TCT AGC AAG CTC CCT AAG CTG ATC AGT
443

CTC ATC CGC ATC ATC TGG GTC AAC TCT CCC CAC TAC AAC ACT CGG GAG AGA CTG

ACC TCG CTC TTC CGA AAG GTG TGC ATA TGC TGA GGG TGG GAT GGA GGG GTT

TAT GAG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F242
TAT ACG CAG TGC TAC CAT CGA TCA CAA TGC ATT CGG CAG AGG ATC TGT GGT TGA
444

AAA GGT AGT GAA GAT TGC CCT TCT GCT CAG AGA CTG AGC TCA GAA GGC TTC TAC

CGG CAT GAT CTG CTT TAG GGT TGC TCA GCA TTG GAG CCT GGG CTG GAC TTT CTG

TTT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F243
TAT ACG CAG TGC TAC CAT CGA TCA CCA GGG TGG CTG CAG AGC TGG GCT GAG
445

GCA GAG TAA GGG GCC ACC TAG GAC AAG GAG TGG GCA TGG AAC CCA GAG GTG

TCG GCC TGG CCA GGG GTG GAG AAC CAG GGG TGG GAT GAG TTT CAG GAG TCA

AAT TAA GGA GTA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F244
TAT ACG CAG TGC TAC CAT CGA TCA CAT GCA GAG AAG GAG TTG GGT GTC AAG GAG
446

GTG GGG ACA GAG GCT GTG GGG ACT CCT TCT GCA GAT GAG CAA TGA GAT CAT

CCG CTT ATG CTG CCA CGC CAT CTC CCT GGA CCG GAT CTT TGA GGG ATA TGT CTC

TTC CAG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F245
TAT ACG CAG TGC TAC CAT CGA TCA CAA GGA GGA CCT GCA AGG CTG CAT TCT CTG
447

TTG TCA CGC TTG GAA AGA TCA CTA CGT ACA GGC TGT GCA GAT GCA CAT CCA GTA

TGA TAC GCC TCC CCT AAC ATC CCA TGT CTC AAC TCC CTT GTC AGC CCT GTA AGA

GGG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F246
TAT ACG CAG TGC TAC CAT CGA TCA CTA ATC TCT CCT CCC TGA TCC CAG GAA CCC
448

TAC CTG GTC TTT TCC TCC TAT CTT GAC CCC ATC GTC TAA AAT TCT ATT ATG TAT

GAA TCT GCT CCT AGA ACT TAG ATG CTC TGT CTC CTG TCT GGT TCT TCT CAC CTT

GAG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F247
TAT ACG CAG TGC TAC CAT CGA TCA CCT TGT CCT TTG GGA TTC AGG AGG GTT GTT
449

TCT CGG GGT CTG ATT TTT CTA GGG CTT TCC CTG AAT GCA GGG CTC CTG GTC TAG

GTT TGC ATA GCC AGA CTG CTC TTG CCT TCC AAT TCA GTC GGC CTT TCT CCT GAC

ACA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F248
TAT ACG CAG TGC TAC CAT CGA TCA CGT TCT CCA GTC GGG GCT GGG TCC TAG ATC
450

AGA CCA GCA TCT TTG CTC AGG TTG ATG CCT TTG TGC AGC GCT GCA AGG ACC TTA

TTG AGG TGG GAA GAC TGA AGA ACC AAA AGC TAA CAG CAG ACC CTC CAG AAT CCC

TCC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F249
TAT ACG CAG TGC TAC CAT CGA TCA CTT TTA GAC ATT TGA AGC TAA ACC AAA TAG
451

CTT AGG ACT TTG GAG TTT GGA AGG AAA GCA GGA ACC CTC ATG CTG TCT TCT TTT

TTA GGT ATG TGA CTG TCA GTA TCA CTT CGC CCG CTG GGA AGA TGG CAA GCA

GGG TCC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F250
TAT ACG CAG TGC TAC CAT CGA TCA CTA TCC TGG ATG TCA AGA ACA CCT GTT GGC
452

ATG AAG ACT ACA ATA AGT GAG GGA ACC ACA GGC TGA TGC CAG GCG TGG GCA

GGG AAG GCA GAT CAG GCA GCC AAG AGT GGG AGG AGT GGC GAG AGT ATG CAA

AGG AAA TGG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F251
TAT ACG CAG TGC TAC CAT CGA TCA CAC TCC CGC AGC GGG GTG CAG CTT TCC TCT
453

GGG ATG AGT GAC CGG AGG GAA CCC GCC TTC CCG GGC ACG TCG CCA GCC TCT

TCC TCT TCT TCC CTA GGC TAT CAA GCG GAC TTA TGA CAA GAA GGC GGT GGA TCT

CTA CAT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F252
TAT ACG CAG TGC TAC CAT CGA TCA CCT GTT CAA TAG CGA GCT GGC CCT GGT GAA
454

CCG TGA ACG GAA CAA GAA ATG GCC AGA CCT GGA GCC CTA CGT GGC CCA GTA

TTC CGG AAA GGC GCG CTG GGT GCA CAT CCT CCG GCG TCG CAT CGA CAG AGT

CAT GAC CGT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F253
TAT ACG CAG TGC TAC CAT CGA TCA CAT GGC TCC TGA GAG GTT CCC CAA AGA GTC
455

TTC AGG ACC AGC ACC TAT GTC GGT GAG GGG AGT GGC AGG TCC AGT TCA GTG

AGG TCA GTC GTG GGT TAA GAT CTG CAG AGG TGG TGT GTG AGG CTG GTA GAA

CAT GCG CCA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F254
TAT ACG CAG TGC TAC CAT CGA TCA CTC ATT TTA GGA ATT TCA TAT CAG GCT GCA
456

GAG GAG GAA TAG GGC CGG TAT CTG GGG TGT TGG GTC AGT AGA AGA TAC AGC

CTC ATG ATC CAG AGA CCT GGA CAG GTG ACA CCC TTA CAG AGC CTG GTC TCT CTG

GAA AGA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F255
TAT ACG CAG TGC TAC CAT CGA TCA CTG AAC ACA CAC ACC CTT TGG GAA TAA AGG
457

TTG AAA GCT GTT GAG GTA GTG ACC TGC CAT CAT GCA GCT GGT GCT GGT AGA GAG

TTA GGC AGC CTG CAG ATA GCT GGA GGC TGG TTT GGG CCT GGT GGA CTA CGC

CCA GGC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F256
TAT ACG CAG TGC TAC CAT CGA TCA CGA CTC CTT TTC TCA TTG CGC CTT GGT CTC
458

TTC CTA TCA CCT TCC TTT TTG ACT CTA CCT TCC TTC TAA GTT ATC AAA ACT CAA

TCC ATC TCC ATA GCA AGC CTT ATG CAT ATT GTT GGG GTA TGT GCG CAT GCG CGC

ATG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F257
TAT ACG CAG TGC TAC CAT CGA TCA CGT AGG TCT CAG GGA GAT GGT GGC CCC
459

TGG AGG AAG GTG GCA GGC CGA CTC CAC CCA CCT GTT CTT CTT CCC CTC AGT

GCC TTG CTG GTG CTC ATT TCC TGC CCC GTA TTG GGA CTG GAA AGG AGA GTG

TGC ACA CCT ATC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F258
TAT ACG CAG TGC TAC CAT CGA TCA CGC AGA TGG TCC AGG CCA TTG ATG AGC TGG
460

TTC GAA AAA CCT TCC AAG AGT GGA CAT CAA GTC TGG ACA AGG ATT GCA TTC GGC

GGT TGG ATA CCC CAT TGC TGC GAA TCA GCC AGG AGA AGG CGG GCA TGC TGG

ATG TCA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F259
TAT ACG CAG TGC TAC CAT CGA TCA CCC ACT CCA CTA TCT GAC TAA ACC AGT GAT
461

GTC GCT GTT CTG CCA CTA GAT GGC CAT AAT GGC TCA GCT AAG TCA CCC AAA CCC

TGT CCC TTT CCT GAC TGT TGG TGG GGC TGT CAG ACC ATG CCT TCT TCT ACA GCT

TCA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F260
TAT ACG CAG TGC TAC CAT CGA TCA CTG CTT GTG GGG CCA AAT GTA GGA GGG GCT
462

TTG CCA GAG AAG TGC TGT GTG GAG ACG GAG TCC CAG GGA CAG ATG AGA CTG

TGA GTG TTT GTC CTG GGC AGC AAA AGG TGG CCC AAC CAG GGG AAG AGG GCA

CAT TCT GGC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F261
TAT ACG CAG TGC TAC CAT CGA TCA CGA GAA AGG AAG CAA AAG AAC CAG GAT GGA
463

GAA GCA GAG CGG AGT GTC AGG TGA GAG GGG TGA GGC AGA GGC ATC AGT GAG

GAG GAC AGG GAT GTT GGG AGC TGC AGG GAT GAG GAA GCC GTT GAG GAG CAG

AGT GTG CAC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F262
TAT ACG CAG TGC TAC CAT CGA TCA CTG GCT AGG TCG AGA GGT TTG AGA AAA GGC
464

TTC GAG TAG TGA GAG TTG TTG GAG ATT GGC TTT GCT GAG CTT CCT GGT TTT GTG

AGT TAT GGC CAA GCA CAG CCA CGT AAA CAT TGC CTC ATA AAT CAA CGA TAA GAT

TCT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F263
TAT ACG CAG TGC TAC CAT CGA TCA CAC AGT TTC TGT AAG GTT CTT GAC TAT TCC
465

GTG CTG GTG GAT TTC AAC TGT GAT TCC CGC ACT TTG TTC CCT TCC CTC TGC AGC

ACT GTG TTT ACT GAA ATT TGA ACA CAG TGT CCC TCT AGT TAG GAT AAA CCA GGA

GAC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F264

TAT ACG CAG TGC TAC CAT CGA TCA CTG CTC TTT GTG TTC AAG CTC CAG TTA AGA
466

TCT TGA AAT AAG CAG ACT GTC CAA ATG TTC TGA TGT TCC TCG GAA TTA CCT GGC

TCT CTT AGT CCT GGA GCC TTC CAG TCC CTA ATA GCA CTG ATT TAA TCC CAC AAG

GGG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F265
TAT ACG CAG TGC TAC CAT CGA TCA CTG GCT TTT ATC ACG TTT TAC GGA CAG AAC
467

AGC TTC ACA GAT TGC ACT TAA CAC AAA CAT ATT CCA GAT GTC ATC CAC ACC CTG

GCT GAA GGA GCT GTA TTA TCA CCC TGT GTG GAG CAG AGT AGA ATT TCC TGA GAG

GAG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F266
TAT ACG CAG TGC TAC CAT CGA TCA CTC TGC TTC ATT TCT CAT CTC GAC ATT TCC
468

CTG TGC TTC CCA GCC TCT CCA CCT GTT CGA TTG TTG CTC TAG TTT ATC CCC TCG

TGC GGT CCC CTC ACT GCC TGG CTG CTC TCT CTC AAA GGT CCC TTC TGA TTC TCT

TTG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F267
TAT ACG CAG TGC TAC CAT CGA TCA CGG AAA TTG ACT ACT GGG AGC GGC TGC TGT
469

TTG AGA CGC CCC ATT ACG TGG TGA ACG TAG CTG AGC GAG CCG AGG ACC TGC

GCA TTC TGC GTG AAA ATC TGC TAC TCG TTG CTA GAG ACT ACA ATA GGT AGG GCT

TCA GTC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F268
TAT ACG CAG TGC TAC CAT CGA TCA CTC TGT CCT CTA CAT CTT TTC CAT CTC AAA
470

AAA AAA AAA TTG TCT CCC ACT CTT ACG GCC CTA TCA TTA TTC CTA CCC CTT GTA

GTT TTA TTT TCT AGC TGC AGG GTG GAG AGT AAA GCC AAC CAG GTC CCC TCC CAC

CCC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F269
TAT ACG CAG TGC TAC CAT CGA TCA CTG GTC TAG AAA TGT TCA CAT TTC TCC ACT
471

GTT TCC TGA AGT GGA GGA AAG ATC TTC AGG GCT TGT CCC CTC TGG ATA CCA GGC

CTC TCT TAT GCA CAG GAT TAT TGC CAT GCT GTC CCC AGA TGA GCA GGC CCT ATT

CAA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F270
TAT ACG CAG TGC TAC CAT CGA TCA CAT GAC GGG GCC TGA CTC TAG GTG CAG ATG
472

ATT GTG AAT GAG TTC AAG GCA TCC ACT CTG ACC ATT GGC TGG CGA GCC CAA GAG

ATG TCA GAG AAG CTG CTG GTA CGC ATT AGT GGC AAA CGG GTA TAC AGG GAC

CTG GAA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F271
TAT ACG CAG TGC TAC CAT CGA TCA CTT GAA GAG GAC CAA AGA GAG CAT CGG GCA
473

GCT GTA CAG CAG AAA TTG ATG AAC CTG CAC CAG GAT GTG GTG ACC ATC ATG ACC

AAC TCC TAT GAG GTC TTC AAG AAT GAT GGT CCT GAG GTA GGG TTC CTG TGG CCA

GGA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F272
TAT ACG CAG TGC TAC CAT CGA TCA CAG TTC CAT CAG GCC ATT CTC TTG CTC CCC
474

GAC CCT TTT TTC TTT TTT ACC AGT GTG TAC TTC TCA AAC ATC GTG CTC TAG TCT

TGG ACT CAG CCA ACC TTC AGT CCT CAC ACC TCT TCC TCC AGG ACT CAG CGT CCT

GCG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F273
TAT ACG CAG TGC TAC CAT CGA TCA CTT CCA TTA AAC CAA CTT GTT TCC TTC CCT
475

GTT GGC TGG TTC TCA CTT CCC AGG ATG TGC CCT GTC TTC CCT GAA AAG TTC TCT

TTT TCC TCC CTC CAT CCC ATC AGA TTC AGC AGC AGT GGA TGC TGT ACA TGA TTC

GGC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F274
TAT ACG CAG TGC TAC CAT CGA TCA CGG ACC GCA TGA TGG AGG ATG CCC TGC
476

GCC TGA ATG TGA AGT GGT CAC TGC TAG AAC TAT CCA AGG CTA TCA ACG GGG ATG

GAA AGA CCA GCC CAA ACC CAC TCT TCC AAG TCC TTG TCA TTT TGA AGA ATG ATC

TGC AAG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F275
TAT ACG CAG TGC TAC CAT CGA TCA CTC TCA AAA CCA TTG CAT CTT ATT TGT CAG
477

TTT CCC TTA ATT CCT AAA CTT GGT CCT TGG CCT TGA CTT TAT CCT ATG GCC TTC

AGA CCT GCT GCT GAA GTG TGT GAC CTT CCC AGT CCT AAG GCT TTT CTC TCC ATC

TCC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F276
TAT ACG CAG TGC TAC CAT CGA TCA CCT GGG TTC CTC ACA GGT GGA ATT CTC ACC
478

CAC TCT GCA GAC TTT GGC AGG TGT GGT CAA TGA CAT TGG CAA CCA CCT CTT TTC

CAC CAT CTC TGT CTT CTG CCA CCT CCC TGA CAT TCT CAC CAA GCG CAA GTT ACA

TCG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F277
TAT ACG CAG TGC TAC CAT CGA TCA CTT GAG ATT AGG TGA CTG ATG CTC ATG GGT
479

TTT GGG ATT TGG GAT GGG AGA TGA GGA AAG ACA AGC TTG GGA CTG GGA CTG

GGA CTG GCC TGT AAG AGG CCT AGA TAC CAA CAG ACA AGA CAT CAC ATC CTA TGA

AGG AGA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F278
TAT ACG CAG TGC TAC CAT CGA TCA CAC TAA GAG TCA CAT TTT CAC TTT CTG CCT
480

ACT CCT TTA CCT TCT AAT GTG CAT GTT TGA GCT GTA TTT CTC TGG GAA GCT GGT

TTT AGA GTG GAA GGT CTG GAG CAG TGG GCA GGG CTC AGG CAG AAG TTG GGT

TGG GGT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F279
TAT ACG CAG TGC TAC CAT CGA TCA CGA AGT CAG TGA GAA GCA TCT TTC TTG GTC
481

CTT TGA AGA GCA AGA TGA GGA CAT CAA GAA GAT CCA GAC CCA AAT CAG CAG CGG

CAT GAC TAA CAA CGC AAG CCT GCT GCA GAA CTA CCT CAA GAC CTG GGA CAT GTA

CCG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F280
TAT ACG CAG TGC TAC CAT CGA TCA CGA GAT CTG GGA GAT CAA CAA GGA CTC CTT
482

CAT TCA TCG CTA CCA GCG CCT CAA CCC TCC TGT CTC TTC TTT TGT TGC CGA CAT

TGC CCG GTG AGT GGT GAG GGT GGA TTG AAA GTC TGT CTG TAG GAG GCA CAG

CAC TGC GAC TTC GAA AGA CGC TCC ACG ATA T

SigFolig1
TGC GTA AGT GCA TCA GTC CAT CAT GCA TCC GTC GCT ACA TGA GTG ACT AGA TGA
483

ATC CGT CAG TCA ATG CCT GGA TGA GTA GAT CGC TGA CTG CAT ACG TGT TCA GTC

AGT CAG TCA GGC ATC TAT ATA CGC AGT GCT ACC ATC GAT CAC

SigRolig1
TAG ATG CCT GAC TGA CTG ACT GAA CAC GTA TGC AGT CAG CGA TCT ACT CAT CCA
484

GGC ATT GAC TGA CGG ATT CAT CTA GTC ACT CAT GTA GCG ACG GAT GCA TGA TGG

ACT GAT GCA CTT ACG CAA TAT CGT GGA GCG TCT TTC GAA GTC

SigFolig2
TGC GTA AGT GCA TCA GTC CAT CAT GAC CTA GCT ACA TGA CTC AGT CCA TAC CTG
485

CGT CAG TCA ATG GAT GGC TAA CTG GAT CCG TCG ATC AGT AGA TGA GTA ACT GAG

TCG CTG CGT CAG TGA CTG TTC AGT CAG TCA GTC AGG CAT CTA TAT ACG CAG TGC

TAC CAT CGA TCA C

SigRolig2
TAG ATG CCT GAC TGA CTG ACT GAA CAG TCA CTG ACG CAG CGA CTC AGT TAC TCA
486

TCT ACT GAT CGA CGG ATC CAG TTA GCC ATC CAT TGA CTG ACG CAG GTA TGG ACT

GAG TCA TGT AGC TAG GTC ATG ATG GAC TGA TGC ACT TAC GCA ATA TCG TGG AGC

GTC TTT CGA AGT C

SigFolig3
TGC GTA AGT GCA TCA GTC CAT CAT GGC ATC AAT GCA TGC CTG AGT AGA TCC GTA
487

ACT GAG TCG CTG TTC AGT CAG TCA GTC AGG CAT CTA TAT ACG CAG TGC TAC CAT

CGA TCA C

SigRolig3
TAG ATG CCT GAC TGA CTG ACT GAA CAG CGA CTC AGT TAC GGA TCT ACT CAG GCA
488

TGC ATT GAT GCC ATG ATG GAC TGA TGC ACT TAC GCA ATA TCG TGG AGC GTC TTT

CGA AGT C

SigFolig4
TGC GTA AGT GCA TCA GTC CAT CAT GAA CTA GAT ACA TAA ATC AAT CAG TGC GTC
489

AAT GAG TAA ATA GGT AAG TAG ATG ACT AGC TCC ATC GAT GCA TCA CTG CGT AGC

TAG CTA CAT GAC TGC ATG TTC AGT CAG TCA GTC AGG CAT CTA TAT ACG CAG TGC

TAC CAT CGA TCA C

SigRolig4
TAG ATG CCT GAC TGA CTG ACT GAA CAT GCA GTC ATG TAG CTA GCT ACG CAG TGA
490

TGC ATC GAT GGA GCT AGT CAT CTA CTT ACC TAT TTA CTC ATT GAC GCA CTG ATT

GAT TTA TGT ATC TAG TTC ATG ATG GAC TGA TGC ACT TAC GCA ATA TCG TGG AGC

GTC TTT CGA AGT C

p53F001
TAT ACG CAG TGC TAC CAT CGA TCA CAA TTC CCA GAA GTA AGA CCA TCT TTG GGA
491

CAA GAG ACA ATG AAG AGA AGT CAG ATG TGG AGG AAC AGA AAA CAA GAG CCA

GGG GCC AGG GCA GGT CAG TCC TAG AGA AAC AAA TGG ACC AAA CTG GGG ACA

AAG AAG GCA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F002
TAT ACG CAG TGC TAC CAT CGA TCA CCG GAG AGC TCC CCA TTC TCC GAG GGG
492

CCC TTA GGA AGC TTG CTG ACA GAG TCA CCC TGA GGG GAA AGT GGG AAA GAA

AAC AGA AAA GCA AGA AGC GGT GAG TAG AAA TTC AGG TGG GAG ACA TTC CCT ACC

ATC CAA GCC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F003
TAT ACG CAG TGC TAC CAT CGA TCA CTG GTG ATC CAG AGG TTG GTT CCC TGA TCT
493

CAT GAT CCA GTC TCT CTT ACT TGG GAT CCA GAA AAT GTG AAC CAC ACT TTC TTC

TCT ATC CTA TTC AAA GTT TAC ACT GGT CTA AAC TCC ATC AGA CAC AGC TGC CTC

TGC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F004
TAT ACG CAG TGC TAC CAT CGA TCA CTG CCC CAG AGT AAC AAC AAA AAA GGG ATG
494

GGG TAA AGC ACA TCT TGC CTG CCT ACC TTC TGT CCT GAA AGA GAG TCT TCT GAG

GGT TTA GTG CGT TCC AGG GGT GGC CTC TGG CGG CTC TGA GAC TCT GCT CGT

GAG ATA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F005
TAT ACG CAG TGC TAC CAT CGA TCA CAG TCA GCC ACA GTC ACT GGG GAA GGC
495

AGG AGA TTA AGA CTT TCA GAT GGA ATT CTG GTA GCC AGC CTA AGA CAC CCA CTG

AAC CCG AAC CCC TGA TTT TCA CAG GGG TGA GCA TCC CAT TCT CTC AGC TCT GAG

GAA GGA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F006
TAT ACG CAG TGC TAC CAT CGA TCA CTC TGG TGT CCA GAG TTG GCT GAC CTG GCT
496

GGC GGT CTC CAC TGA TAG AGC CAT CAG TCC GAT TAC CAC GGT TAC GGC GGC

GGC GGC TGC GAT TAC GAC GTT GAG GTC TTG ATT CAT CTT CTG TAG GGT AGG AAA

AAA CCA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F007
TAT ACG CAG TGC TAC CAT CGA TCA CAG TCA CAC ATC CAA CCT CCC ACC CTC GAT
497

TCC TAC CCA TCT CTA ACT TTC CAG ACC CCA GGC ACA CCC TCA CCC AGG CCA TTC

TCT GTC ATG TTG GGC CCA TCT GAT TCC AGG CCT CCA TCC ATG ACG GTC CTG TCT

TCA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F008
TAT ACG CAG TGC TAC CAT CGA TCA CCT CTG GTT CAG ACG TGT CCA ATA GGC TGT
498

AGG GAT TAC TGT CTG GAT CCT TCA GCA CTG GTC AGA GGA AGA AGA GGA GGA

GTT GGC AGT CAG GTG CCC ATC ATC TTT CCT TTT GGC CCA TAT TCA TGA ACC CAG

CTG TCT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F009
TAT ACG CAG TGC TAC CAT CGA TCA CAT CCC TGT CGC CAG GCC CAG CTC GGT TGG
499

GCT CCT CTC TCT TCT CTG ACT CAG TCT CTG AAG CTG TAG ACA CAT CTG AGC TGG

GGC CTG AAG AAC ACA ATG GGA TTT ATT CAC GGC CAT TCA TCC CAC CTC AGG ATC

CAT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F010
TAT ACG CAG TGC TAC CAT CGA TCA CAA ACT GGA AGA ATT CTC CTT CTC ACT CAC
500

AAG CCT CCC AGC CAA TCA ATC ACT TTT TCA TCA TCT CCA TTC CAG ATT TCC CCA

AAC TTC CAT CCA GAC CTC TGC CTC TAC CCA CAA GCC CTG CCA CTC CTT GCC TCC

TGT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F011
TAT ACG CAG TGC TAC CAT CGA TCA CAT AGG TTC GAG TCG CAT GGA GGG AGG
501

AGG AGG AGC TCT CAT CAG TGC TAT ATC CAG CCT TGT CGC TGC CAC CGC TGC

CCC GCC CAC TCC CAG GAG GGC GAA AGC CCA GCC CAA TCT GCC GAA GCT GCT

CAT CAA TTT GTA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F012
TAT ACG CAG TGC TAC CAT CGA TCA CCC TCT CCA AGC GAA GCT GCT CTA CCT CCT
502

GGT TGG GTA AAA GAT GGA AGA AGG GGA AGG AGA AAT AAG ATC AGT GCC TTG CTT

CAT GCT TCT GCA CCC TGA CTG TCC CTC TAT ATC TAA CCT CTC AGG AAT GCA GGG

AGA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F013
TAT ACG CAG TGC TAC CAT CGA TCA CAC AGC AGT CTT AGT TCC CTT TTC ACA CCC
503

AGC ATT TTT CTC TCC CGG CCT GGG TAC CTG CAG GTA GGA GAG GTG ATA CTC CAG

CAA AGC CTG GGC ATT GCT GAT GTT CTC TCG GGT GCC AAC AAA AAT GAA GGG AAC

CAT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F014
TAT ACG CAG TGC TAC CAT CGA TCA CCC CTA GAG AAC AGA GAG CAG AAA CGA TGG
504

ATC AGA AAG GAA AAT GAA ATG GAA GGA TTA ACG CCG CCC AGT CTC CTA GGG

GAG CCT GCC CAG TGG GAT CAT TCT GGG CTC TAA ATC TAC TAG TGT TAA ACA ACT

TTC CGT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F015
TAT ACG CAG TGC TAC CAT CGA TCA CCT CCA CCC TCT CAA AGA ACA ATC CCA GCC
505

CTC AGA GGA CAA TCC CAT TTA CTT CTG GAA ACC AAT CTC TAT GCC CAC TTG CCC

CGT ACC CAT ACC TCC TCC CTG GGG TTC TTC TTG TCA TTA TCA CCT TCC ACT CGA

ACC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F016
TAT ACG CAG TGC TAC CAT CGA TCA CTC ACT CCC ATT ACA GAC TGT TTC TCT GTC
506

CTT GTG AAA CTG GGA GTC CAC AGG GAG GTG CCA ATC CTG GAT AAA AGC TCA GAA

TCA CTG TAG AGA ATC TTG ATT CTG ACC TCT AAC TGT ATA TAC ACA CCA CCA ACT

CAC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F017
TAT ACG CAG TGC TAC CAT CGA TCA CTT TTC GGG CCT GCT GGA TGT TGG CAC CGT
507

GAG TCC CAA TTG CCA GTC CCA TCA GGT CCT CTC GCA CTG TGA ACT CCT CTT GGA

AGG CTG CTG CCA ACT GCT TGC TTG TCT GAA AGG AAA AGT CAC TGT AGG AAT CAT

GGT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F018
TAT ACG CAG TGC TAC CAT CGA TCA CGT GAG GGG TTA AGA AAA ACA GAA AAC ATG
508

CCT CTA GAA CAC CAG GCT TTC ACT TCA TTT CTC AAT CAG TCC TTA GAG GAA CCC

CTG GGT TCA GGG GCA GGA AGA CAG GAA AGG TTT TAG AGG AGT GAA TCC CAT

GTT GTA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F019
TAT ACG CAG TGC TAC CAT CGA TCA CCA GGG GCT TCT GTG GTT GAC TGG GAG GAA
509

ACC AGT GGG AAA GTC ATT AAA ACT GGC TTG GAA TAA ACA CTA AGG GAG AGC CAG

GGA CTA GAA ATC AGG ATG CCT AAG TCC CGG GAA AAG GGT TAA GAA ATG AAG CTA

GGA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F020
TAT ACG CAG TGC TAC CAT CGA TCA CTT CAG GCA GAA TAA ACT CAC CAG AAT GAA
510

GAG CTC ACT GTT TGT GAT GTT GAG AAA GAT GCA GTT GGC TCC CAG GGC TTT CTT

GAA CTC TTT ATG GAC GTT TTC ATT GGA GCA GCT GCA GAG GAA AAA AGT ACT CAG

CGG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F021
TAT ACG CAG TGC TAC CAT CGA TCA CGA AGA GGG ACC ACA GTT AAA GAA CAA GGA
511

TGG GAA AAT AAA GAA ACT AAG TGT CTT CTT AGC AAG AAG GGC TCT AGT AGA GGG

CCA GGG GAA CAA CAG AGA AAA CTG CAA AGT GGG AGT GAT GCC TGG GAG AAA

TAA AGC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F022
TAT ACG CAG TGC TAC CAT CGA TCA CGA CAT AGA ACT GGC ACA TGG TAA AAA AGG
512

AGA AGT TGT GGA TGA CCT GAG CAA CTT TCT ATG AAT AAC ATA TTT GTT TCA GTA

GTT CTC CAA AGG CAT TCC CAG TCA CTC ACA TCT CCC TTC ATC ATC CGC ACC CGG

GCC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F023
TAT ACG CAG TGC TAC CAT CGA TCA CGT TAC CAT AAA TGG AAG GCA AAT CGC CTG
513

AAA CTG GGG ATG TTC CTG AGA GTT TGG CTG ATC ACA ACA GTG ATA AAA GGG GAA

CTG TTG CAT TTT ACA TTT CCA TAG GCT ACA TGG CTG GAT ATG GCT TAG TTG GGG

AGG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F024
TAT ACG CAG TGC TAC CAT CGA TCA CAG GAA CAT GAA CCA ACC AGA GAA AAG CCT
514

AGA ATC CTG GGG AAA AGT GAC ATT TAG AAA GGT GTA CTC CAC AGC CCT TAC CTC

CAC TTC ATC CCC TTC TGT GAT CTC CTT ATT ATA GTC AGC TGG AGG TGG TAG CCG

GAC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F025
TAT ACG CAG TGC TAC CAT CGA TCA CAA TAT GAA AAA GCT ACT CTG ACT GAT ATG
515

GTT GCC CAT TTC TTT ACA AAA GGG TTA TTG CCT CAG CTG GGC GAG TTC CAC CTA

TCT GGA TAA TTA GAG GCT AAA CCC CAG GCC CTG CTC CAT AGT AGT GTC TTC CAT

CTC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F026
TAT ACG CAG TGC TAC CAT CGA TCA CCT TGA GTT TTA GAT AGG GAC ACA AGT GGT
516

GTA ATG AGA CAG CCT GAA AGG CTG CAC TGG GGA AAG AAT ATG GGC AGT CAA

ACA GAG AAA GGA AAT GAT ATT GTC ATG GAG AGG TGT ATG TAC CAC CTG CAT CAG

AAC AGC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F027
TAT ACG CAG TGC TAC CAT CGA TCA CTG GAA ACC AAT GAT CTA CTC AGG CTC CCA
517

CAG GGG CCT CCC TAT CTG TCT GCC TCC TGT TGT GCT AAG CAG CCT GCC AGA GTT

CAA TTA AAA TAC CAA CAA CAT TCA GCA AGG CTA AGA ATA TCC CAT GAG ACA GTG

TGA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F028
TAT ACG CAG TGC TAC CAT CGA TCA CGC TGT CCT GGA ACA CCT AGA GGA CAT GAG
518

GAT CTG TCG AGA GAG AAG GGC CTA CCT CCG CAA TAC AGC AAG AGA TCT GAG

CAG ACT CGC TGC CCT GAT GCA TGG GTC CTA AGA CAA TAA TCG GAA CAC TAG ACA

AGT TCA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F029
TAT ACG CAG TGC TAC CAT CGA TCA CAA AGG ATA ATA AAA TTT ATG GAA GAC TCC
519

AAT CTT GAT ATT TTG ACA AGA GCA AGA GGG ATG ACT GAA CAG CTG ATC CAC CTC

CTG TGG TCT GTG TCC TCA CTA AGG AGG CCA AAG CCA TGC ATT CAC CCC TAT GCT

CCC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F030
TAT ACG CAG TGC TAC CAT CGA TCA CAG AAC CTG AGT TAT GAG CAT GTT CAT TCA
520

GGT TTA ATG CCA ACT CCT ACT GTT CTC CAT TCA AGT CTT CTG AGG CAC TTT TAC

TTC TAC TTG CCT GGG TCT CTC TTC CCT TTT GGG TTC TTC CCT TAG CTC CTG CTC

AAG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F031
TAT ACG CAG TGC TAC CAT CGA TCA CTA CTG CTT TTG TGG AGA GAT TTC TAC TCC
521

TAG ATA TAT TCA AAT CGG CTA TGC CTA TGG TGT AAT CCA ATT CTC CTC CAC CAT

GTT AGG CTT GGC CCC AGG GTG AGC AGC AAT ATT ACC CTA GTT TTC CCA GCT ACC

ATG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F032
TAT ACG CAG TGC TAC CAT CGA TCA CCC TTC AAT TAG TCT CCA AAA CTA GCT CTG
522

TCC CCA ACT TCA TAT TCC AAT CCT GTC TCT GCT CCT AAA TTC CAC CCA TTC ATC

CCA TAC TAG TTT TTT TCT CCA AGG GTA TCT GTG GTG GAA GTG AGC CCC AAG TTC

TGC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F033
TAT ACG CAG TGC TAC CAT CGA TCA CCA GTC TAC GCT CAT TGT GAA AAT AAG CAG
523

GTG AAG GCA AGA GCA GGT GGG TGG AAT GTA TTA GGG AGC CAC CTA GAT CCA

GAG GAT AAC TTG ATT CAG TGG CCA TAG CAT CAC CTG GCT TTG GCT TCT GGA GCC

TGG CAC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F034
TAT ACG CAG TGC TAC CAT CGA TCA CTT CTA GGA GAA GAG CAG GAA CAG TCT GAG
524

TAA AAA CCA AGT ACT GAA GCT GAG GAT GCC ATG TTG ATT AAG AAA GGA ATG GGG

ATT AGG ATA TCC AGA TGA GGT TTG GAG TAG GAA GAT TGC GCC CTG GAC GGT GAA

TAC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F035
TAT ACG CAG TGC TAC CAT CGA TCA CAA CTG ACA ACA GGG CCC AAT TCT GAG AGA
525

GGA GAA AGG GAC CTT CCC AAA TGT ACA ATT CCA GAA GCA GTG TAA ATA CTC CCT

GAC CCA TCC CAA GTT TGC TGA GTC ATG AAT ATT AAA TCA GCA GCA AGG TCA AAA

CGG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F036
TAT ACG CAG TGC TAC CAT CGA TCA CGT ATC TGT AGG GTA CTG AGA GTG GAA GAT
526

TAG CCT GAA TTT AGG GAT TTG TGG TTT TTA ACT CAG GAA CAG GGC AGA AAA TGC

TAA CCT GCC CAG ATC CAC ATT ACC ACA TTA CCT GGC TTT CAG CTC AAC CCC AAA

CTC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F037
TAT ACG CAG TGC TAC CAT CGA TCA CAC TCT CTG AAT CAC AGT ATC CTG GTC CCC
527

ATC TAT TCA AGT CTA TCA CTG CCA CAG CAC ACA GGA GAA AGG GAA AAG ATT CAG

AGA GAC TTT CTG CTG GAC ACT TAT ATG TGG GAC AGA AAA CGT GTC CCT TCC CAG

CCA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F038
TAT ACG CAG TGC TAC CAT CGA TCA CAT CCA CTC TTA ACA AGT GAT GCC ATG GGA
528

AGG TAT CTC TTC CCC AGC TCC CCA GGG TGA AAT CAA AGA ATG AGA CAG AAA TCA

GGA GAG TCT CTA GGA CAA TTC CAG GCA TCA AAT CCA AGA GGT TCA GAA TAC CAA

CAG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F039
TAT ACG CAG TGC TAC CAT CGA TCA CTG ACT ATG AAG CTT ACA TGC CTC AAG ACG
529

AAG AAA CTA TAT GAA TGA CCT GAA GGT ATC TGA GGT CTA AGT GAG ACC TCA AAT

CCA TTC CTG AGT AGA ATG GTA GAG CAG TGG GTC TTG AGA TCA AGG TGT GGT

GGG GAG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F040
TAT ACG CAG TGC TAC CAT CGA TCA CGA GAC TTG CGA AAG ATG CCA CCT TAT GGC
530

TGG CTG GGA AGC AAC TTG ACT GAT CCA ACC CAG GGT TCC TGA GGT CAA AGA

TGC CAA ACG TCG TAA ATG GAT CTG ACC ATT TCT CCT TGG CTT GGG AAC CGG AAT

ATA AGA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F041
TAT ACG CAG TGC TAC CAT CGA TCA CGA AAG TGG GGT GAG TGT TCC AGA GCC AGT
531

AGG CAG AGG CCT CTC TGA GGA AGA TGA AAG GAT CTT TTA CGG GAC AGA GGG

CCT TCC CAA AGG GAC CGT GTG GAA GAA AGA CAA TTC TCC ATG TGC TTG GAT CGT

GGG GAA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F042
TAT ACG CAG TGC TAC CAT CGA TCA CAT GTG ATT AAG GTC TAA GGT ATG TCT TCC
532

ACC AGA CAA CGG ACA CAG TCA ATT AGA AGC TGG GTA AAG GGG TCT CTC CTG

CGG AGC GGG GAG CGC CAA GCC AGG GAC AAT AAT GGC CTG AAG TTC ATT CTC

CCG GAG ATT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F043
TAT ACG CAG TGC TAC CAT CGA TCA CTA GAA GCA GGT GCA GGT GCC TTA GAG
533

GGG TCA AAA ATA AGA GGA ACA GGG TTC ACT CTA AGC GGT CTC CCA GGG AAG

GCT GCG GGT TGG AGC AAG GGT CCA AGA TTC TAA GGG CCA GGA CTC AGC TCC

AGA AGC TCG ATC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F044
TAT ACG CAG TGC TAC CAT CGA TCA CAC AGC GCT CAG GCC ACA CCC ACT CCG
534

CCG CCG CTG GCC CCA CCG GGC TTG GAC AAG TTA GGG ATG GGC ATG CGC CTG

AAC GAC TGC TTG GTC ACT TTC CCC ACC TTT GTC TTT CTT GGT GCG CAC GCT CCA

AAA AGA AAA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F045
TAT ACG CAG TGC TAC CAT CGA TCA CAA AAA GAA GAG GAG TCG CCG CAA GGA AAA
535

AGG TGC TTG GTG CGC ACG CGC TGA GCT TTA CCC TCT TCG CGT ATG CAT CGC

CGA TAT TTT AAG AAT CTG TAA CTC TCT ACT GTA GTA GAT TTA ACA GTC ATG GCT

CTT ACC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F046
TAT ACG CAG TGC TAC CAT CGA TCA CGA CCC AAC ACC ACA TCA GGA CAT GTA ATT
536

CTT ATT TAT TTT TCA CCC TCA ACA AGG AAG AAA GGT CTC TCC CTC AAT TCT GCT

CTT CCA ATA CTT GAG GAT AGG CAC CCC TAA CCC TCC TTC CTC CAG GGA GGC CTC

AGC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F047
TAT ACG CAG TGC TAC CAT CGA TCA CTC AGT GTC TGT GGA CGT AGT CTC TGA AGA
537

GTG CTT CAG CTG ATG GGG AAG GAG AAA CTC AAG ACA GAG ATC CTC CTA GGG

ATG GCG TCA CTT TCC TGC CAA CTT TCT CGT TGC CTC TCC TTG AAA GCA GAA GAA

GTG CCA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F048
TAT ACG CAG TGC TAC CAT CGA TCA CCC CTC AGC TTC CGT CAG ATC TTG GGC TCC
538

TAG GGC CTT GTA CAA GTC CAT GGC CCT CTG GTT CCA GTC CAG GAC GGC CAG

GCG GAA TTG GGA GCA GCC CTT ATC CAA GGC CAC CTG TGG GAG AAG ACA ACA

CTA ACT TTT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F049
TAT ACG CAG TGC TAC CAT CGA TCA CCT CCA TAT ACC CTT GCT TCT TCA GGT CCT
539

CAC TTG TCG CCC CAC CCA TCT CCT CAC CTC AGC CAC CTT TTT GAT TAT TTT GGA

ACC AAT CCC TTG ACC TGT TGT GGA GAG AAA GAG GCA AAA AAT AGC TAT TGT TTG

AGC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F050
TAT ACG CAG TGC TAC CAT CGA TCA CGA AGG GGA TCA GAA AAT GAC ACC GGC
540

TGG GCT CTG GGG ACA GGG GAT AAC AGT GGG GTC TGT GGG GTG CTT TGC TCC

CAC CCC AGC CTC AGC TTC TGC CCA GTA CCC CGA TAT TCC GGC ATC ACA TAG ATA

TCC TCC AGA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F051
TAT ACG CAG TGC TAC CAT CGA TCA CAA ATG GTG CGT CCC TTC CAT GTA CTG TAG
541

ATG AAA TAG TAT ATC CCA TAG CCC ACC ACG CAG GGC CCT GAG AGA GAG AAA

AGG GGA GTA AGG CTT CTG GAA GCC TGT GGG GAG ACC TCT GAG GCC GGC TGG

AGA GGT GGA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F052
TAT ACG CAG TGC TAC CAT CGA TCA CTT CTA AGG GCC AGG TGC TCT TAC CCA GTA
542

GCT TCC CGG GCG CTG GAA GAA TCT CTG CTA CCA AAC AGT GAT AGA AAG GAT TGT

CTC CAA AGC CAT CTG CTC TCA GGG CTG CCG AGA TTG GAG TTG TGA CAA AGA GAT

AGA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F053
TAT ACG CAG TGC TAC CAT CGA TCA CTG CAG TCT TCA CCC GAA TCA GCC TCA GGA
543

TAT CTC CAC AGT CTC CCT CCT TGG CCT CTC GGA TCC GCA CGG AAG CCA TCC GGA

TCC CCG CTG TCT GGG ACC AAA GTC CCA GGG CCT CGC AAA CGG CAA CTA GAC

CCC TTA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F054
TAT ACG CAG TGC TAC CAT CGA TCA CGC AGT GCT TTT TAA ATT GAC ATA TGC AGT
544

GAT AAC CTG CTT TAG CCT CAG GCT CAC TCA CCC GCC CAG ACC CTG GGT AAG CCT

TAA GAC CCT CAG CTC TGA AAG CTG TTT CCT GCA GCT CTT GAG TAG CAT GAA GTG

TTA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F055
TAT ACG CAG TGC TAC CAT CGA TCA CTT TAG GAT TCC ATT ATC TCA TTT CTA GTC
545

CTG ATA CAG GAT GCT ACT TGG GAC GCA GGG GAG GAC TGT TTC TAG ACC TCA

GGC CTG TGA ATG CAG GCT CCC CGA GTG GAC AGA AAT CTT GGA GGA CCT AGA

TCA GGC CCT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F056
TAT ACG CAG TGC TAC CAT CGA TCA CGA GGA GGA GAG GGG AGA TGG AAT ATC
546

CTC TCC CAG TTC AGA AAC TTT CTC GGC AGT GGA GGA TGA TAG TGG AGG GGA CTC

TGT CCT TCA CCC CAT TGA TCC CCA GAG GGG TGA TAG CTG AGT CTT GTG ACT GGG

CCC CTG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F057
TAT ACG CAG TGC TAC CAT CGA TCA CGC CCT GAG ACC TGT TCT CCC CAC CCA GGT
547

GCA GGA GCG GGA CAG GGC ACT CAG CTC ATG CAG TCT TCC CTT CTC TCC TCT

GGC CCT GTA GCA GGG CCT CTC CCT CTG TCT GTC TCT GAC ATG TCC CTA CTC AGC

TTT GTT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F058
TAT ACG CAG TGC TAC CAT CGA TCA CGT TTT CTC TTT CTG ATA GAG TGC CCA CGA
548

CCC TCC GGC TGT CCA CCT CAG CAA TGG CCC AGG ACA AGA GCC TAT CGC TGT

CAT GAC CTT TGA CCT CAC CAA GAT CAC AAA GTA TGG GGT TGG CCT AGC CCT TGA

CCC AGT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F059
TAT ACG CAG TGC TAC CAT CGA TCA CAA ACC CAC ACT GGT TCT CAA AGG ACA CAT
549

GAC ATA CAC AAT CTT TCC TTC TGT GTC CTT CCA GAA CCT CCT CCT CCT TTG AGG

TTC GAA CCT GGG ACC CAG AGG GAG TGA TTT TTT ATG GGG ATA CCA ACC CTA AGG

ATG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F060
TAT ACG CAG TGC TAC CAT CGA TCA CCT GGT TTA TGC TGG GAC TTC GAG ACG GCA
550

GGC CTG AGA TCC AAC TGC ACA ATC ACT GGG CCC AGC TTA CGG TGG GTG CTG

GAC CAC GGC TGG ATG ATG GGA GAT GGC ACC AGG TAA GCT AGC TCT GGT CCT

CAG GGG AGG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F061
TAT ACG CAG TGC TAC CAT CGA TCA CGC TGC TCT TCC CCG CTT CCA ACC TTC GGT
551

TGC CGG TAA CTA CAC CCC AGG GGT GGA ACC CTA GCC AAG ACT TGG TAA AGC

ACT GCT GGG TGG CTG GCC GTG GGA ATC TAA GTC CAC ACT TTT AGG GAG AAG

GGA AGG GTT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F062
TAT ACG CAG TGC TAC CAT CGA TCA CGC CAA ATG CTC AGA GGG GAG TCA ACT GAG
552

GGC AGG GAG GTC GGG ACT GCG GCT CCG ATG CCC TGA TTT CTA CAT CCC CGT

ATC TTA TCT CTG TCA CAC TCC AGC TGG TTC CTG CCC TGG ATG GCT GCC TGC GCC

GGG ATT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F063
TAT ACG CAG TGC TAC CAT CGA TCA CTC TAC CAC TGG CCC CTT TCC TCC TTG AGA
553

CCC CAG CTT TGA GGC CTC AGG ATA ATC ATT TCT CCC CAC AGA CAT TCC CCA GCC

TCA TGC AGA GCC CTG GGC CTT CTC TTT GGA CCT GGG ACT CAA GCA GGC AGC

AGG CTC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F064
TAT ACG CAG TGC TAC CAT CGA TCA CAC AGT GGG GCA TTG CCT GTA TTC AGT GGA
554

GCC TGG AGC AAT GAG GGA AGA GGG GAG TCC AAC ATG TCA ATA TTA GGA AGG

TTT CCA GCC CAG GGA ACA TAA CAA GAC TGG CTC CAC AGA ATT GTT TTT CAT TAA

TAA TTT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F065
TAT ACG CAG TGC TAC CAT CGA TCA CGG CTT ATG GAT GGC ACT CAG GTG GGT GGT
555

AGG GGC GAG GGA CAT ATC TTG AAG CTC CCC ACA GCA AGC AAA CAG TTT TGA CTT

AGA CTG CAT ATT TAC TTG GGG CAG GTG TGG TTT CAA AAA GGG TCA AGC CAA AAA

AAA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F066
TAT ACG CAG TGC TAC CAT CGA TCA CTG GGG CAG GAT TTA AGT GGT GAG AAT GGC
556

CAG TAG GTG GAG GCA TAG CGA AGA GGC AGA ATT AAG GCA GCT AGG GGT GAG

GCC ACA GGC AGT AGG CCC GGC TCA TTC TTC CCT CTC TCT CTA CCG TCC CTT TCC

CAC ACA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F067
TAT ACG CAG TGC TAC CAT CGA TCA CTC TGC AGA AGG TGG TGT TGT CTT CTG GGT
557

CGG GGC CAG GGC TGG ATC TGC CCC TGG TCT TGG GAC TCC CTC TTC AGC TGA

AGC TGA GTA TGT CCA GGG TGG TCT TGA GCC AAG GGT CGA AGA TGA AGG CCC

TTG CCC TGC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F068
TAT ACG CAG TGC TAC CAT CGA TCA CCA GTG GAA GGT AGT GCT TTT GCA AAC TCA
558

GGT TGG AGG AGT GGA AAA GTG GGG AGA AGA TTC TGG ATC CGA GCC ACC TTA

ATG CTC TAA TGC CAC CTT TGC ACT ACC TCC CTC TAG GAG AAG ACT CTT CCA CCT

CTT TTT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F069

TAT ACG CAG TGC TAC CAT CGA TCA CGG AAA TGA GGA CTT CTT AGG CTG AGA TCC
559

CAG CAA AAA CCC CGA CAG ACA GAC ATA CTC TGG ATA CAG GTC ACT TTC TGG AAG

CCA GGA CCC ACC TGG TTC CGC AGA AGC TAC TCC CTT TGA GAC CAG TTT CCT GCC

TCT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F070
TAT ACG CAG TGC TAC CAT CGA TCA CCG TGA GAG GGT CAC AGA GAG ATG TGG
560

GTA GGG GTT CTG GAA AAA GGG GTG GAG GCA GGG AGA AAG AAA AAG GGA GGA

GAG CAG AGG GAG AAA GTC GCG TCT CTC TCT CTC TCT CCT AGT CTT GGT CCA CTT

GCT GCC TTC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F071
TAT ACG CAG TGC TAC CAT CGA TCA CCA GAC AAA TTT GTC TCT ACT CCA TTA AGC
561

AAG AAC TGG CTT GTG CTG GTC CCA GCT GGG AAA AAC AGA CAG ATT TGG GAA TAT

CTC TCC CCT CAA AGA ATA CGG TGA CCC AGC TCT CAA CCA CAG ACC TCT AGA GAA

TGC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F072
TAT ACG CAG TGC TAC CAT CGA TCA CTG CCC AGC ATC CCT CCC TGG GGA ACC CTC
562

GTA TCC AAG CCC ATA GCC CCT ACC CCT CAG CTC CCA GTC TTC TGC CCT CTT AGG

AAC CCT TGT GTC CTA GCC CAA AGC TCC CAG CCT ACT AAG GAG CCC TGG GGC TAA

AGT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F073
TAT ACG CAG TGC TAC CAT CGA TCA CTT AAA AAG AAC CAT TAG GTT AAA GCA TCC
563

TCC ATC TCC AGA CCC CAT GAG AAT CCC AGT GTC CAA GAC TCC TAA TTT CTT ACA

AGA ATC TTT TCA CCA GTA ACC CAG GCA GCA GGC TGA GTC ACT CAC TGG AGC TGC

TGT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F074
TAT ACG CAG TGC TAC CAT CGA TCA CCC CTT CTG CAG AGA TCA GCC GGG ACC TAA
564

ATG ACT CCA GGG TAA GGA GGC CCT GGG GAG TCT GTA GGG TAG AAG ACA CAA

ATC CCA AGC CCC ACC TTC TAC CTC TCC TAA TCT TGA CCC TAT AGA CCC TCT CAG

GAT CTA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F075
TAT ACG CAG TGC TAC CAT CGA TCA CTT GTT CCG AGT GCC AAT ATC CTG ACC TTT
565

TGT CCC TGG AAT CTC CTC GGA GCT GGC TTT TCT CTC CTC CCT CCA CCC CAC CTT

CTC CAG GAC ACT GTG CCA CCA ACC TCT CTT TCC CCA GCC TGC CCT AGA TTC CAG

GAT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F076
TAT ACG CAG TGC TAC CAT CGA TCA CAT GTT GTT CTT ATT AAT CTC ACA CAG TTC
566

CTG ACA TAT TAG TTC TCA CAT AGT TCC TGA CAT ACT AGT CTG AGC CTG CTG GGT

ATC AAA GAT CTG GGA GGG CGT GTG ACA TGG ATG AAC CAT GGA GAG GCT GAG

ATG GAG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F077
TAT ACG CAG TGC TAC CAT CGA TCA CTG TGA AGG CTT TCA TGC CCT GGT TTC TTT
567

CTG GAG ATG CTG GTG GGA AAG GTG GGC TCC AGG AAA AGG CTG GAG GTT TGT

AAC AGC TCT TCT CCT GGA AAT TTT TCA GAT CTC CCC TCT CTC AGC GTG TTT CCC

TCA GAA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F078
TAT ACG CAG TGC TAC CAT CGA TCA CTA CAG TGC AGA GAA GAC ATT TCT TTC TTT
568

CCA AGT GAA CAT GGG GTA CAA GGG TAG AGA CTG AGC CGG GAA TCC GGA GGC

CAC CCC TGG CTG TGT TTG CTA AGC TTT ACG TTT TGA GGA TAG GAC CAG GTT TCA

TCT TTA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F079
TAT ACG CAG TGC TAC CAT CGA TCA CAA ACA GAG GCT GTA TCA CTT CCG TGC TGC
569

TTG CCT GAG GAA GGA AAG AGC AAG TTG CCT CTG AGG TCC CTT GCA GAA GGA

GCC TTT CCA TGT TCA TGC CAA TTT AAC CAG GCA CTA TCT TTG GTA GCT CTA TGG

GTT TCT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F080
TAT ACG CAG TGC TAC CAT CGA TCA CAC CAC ATC CAC TAG ATG AGA TAG GAG GTC
570

AAA AAT ATC CAG CCA TTC TCT AGG GTC TAC AAG GAC CCT TTA TGG TTT GGC CCC

TGC CTA GGT CTT CAA TCT CTT GCT GTA TTC ATC TTG TCT TTG CTC ATT AAA GCC

CAG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F081
TAT ACG CAG TGC TAC CAT CGA TCA CTT AAT CTT TCT TCA AAT CTT GCC GGT TTT
571

CCT TGG AGA GGT TTT TCT TCT CCT TTC AAC TGT CAC CTT AAA TCC TTA GCC CAG

TTT CTC AAC ACC TCA CAT CCA AAC TGC CGT CCA CAT CCT CAA ACC CAC TCC CTC

CAG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F082
TAT ACG CAG TGC TAC CAT CGA TCA CAG AGA ACC GAA GTA GAC CTG GCT GCA
572

GGG CTG GGA TCT CAG GGC AGT GCT GGG GAC TGG GAA TGT GGT GTT GAG AGG

GCT TCA GGG AGA CGT GAT GGG AAG ATG GGG AAG CCT CTG CAC GTG GAC AGA

GCC AGG TGG TGT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F083
TAT ACG CAG TGC TAC CAT CGA TCA CTG GCC TTT ATC CAT CTC TGC GGG GCC TGT
573

CCT AGC TTC CCT CCT GGC TCG GCC AGC CTC CCG GCT GGT CTC TTC GCT CTC TTT

CTT GAT CTC TAG CTC TTT ATT CCT CTG ACA TTC TGC CCC ATC TGC TCC CGG ACT

CTT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F084
TAT ACG CAG TGC TAC CAT CGA TCA CGC TGC TGA TAG TAT GAG TTT TAC CGA GGC
574

TGC AGG TTT TGC TCC CAT GTC GGT GAC GGA GGG AGG AGT GGT CGC TGT GGT

GAT TTG TGT GCA TCA GCC AGC CAG GTG TCT GTG ACA GTC GGA TGA CTT GGA AGC

CTC CCC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F085
TAT ACG CAG TGC TAC CAT CGA TCA CTC TGG TGA CCG GCA CAG GTG CAG GTG
575

AGG GGT GGA ATT CTT CCA AGA GGG ATG GTC AAG CTG GGA CGT TGA GAC ACA

GGG GAC AGA GGA CAC TGT GTG ACA CGA TTT ACA ATC TTT CCA CAC TGG GCA CCG

TCC CCA TCA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F086
TAT ACG CAG TGC TAC CAT CGA TCA CTC CAC CCA TTC GGG GCC TAC ACG AAG TGG
576

GTC CCA TGC AAT CCA TTC CCT CAG GGA ACT CAA ACT CCA GCC CCT GGG ATG AGA

AGA ATC CAG CAA TGC TTG GGA GAG CCA GAG GAC TTC ATG GAA GAA GTG TCC TCT

GAG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F087
TAT ACG CAG TGC TAC CAT CGA TCA CAG CCA TGG AGA GAT CCC ACC AAG GGA
577

AGG CTG TGG GAG ATT CTG CCT TTC CTC CCT GCC TCT GCC CAG GGT GCT GGG

TGT GAA CTG AGG GTG GGG TGA CTG TTG AAG GTT CTA ACA AGC CGT CTC TGA

GAG ATT TGT AGC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F088
TAT ACG CAG TGC TAC CAT CGA TCA CAG GCT AGT GTT AGG TCT TTC ATT TCA GGA
578

ACT GTG TTC AAA GTT TGG CTT CTG AAG GGC ACC AGG AGA GAG ATG TTG CTA TTC

AAA TCT GAG GGT CCA GTC TCT GCG GGG TGG TAT GAG GGT TTG CTT GTG AAT GGT

GGC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F089
TAT ACG CAG TGC TAC CAT CGA TCA CGT ACC CGC TTT AAA AGG CAC CAT GCT AGC
579

ACA GCT TTA AGC ATG AGT ACG AAT GCA GAG GTA ACA GAT GTG TGC CTT GTC AGG

ACT ATG CAT GGT TGA GAA GTT GGA AAT GTA ATT GGA GGC AAA ATA ACA GAC CTC

CAC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F090
TAT ACG CAG TGC TAC CAT CGA TCA CCT GGG AGT CAT GGC TAG AAG CCA GAC ACA
580

ACT GCC TGT TTC CAG TTT GTC TCA TTT TGC CTC CAG AGG AAG GCT CTA AGA CAT

CCC TGT GGC TCT GTG ATC AGT CCC AGT GCA GAA CTT CAG AGT GGG TAG AGG

GGT GTG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F091
TAT ACG CAG TGC TAC CAT CGA TCA CGG GGA TAG TTG AGG TTA TGG TGG GAA CCT
581

TGG GCC CTG CTG ACC CTG TTT CCT CCT CCC TAG CCT TTA TCC TCC TCT TCT ACC

TCG TTT TTT ATG GGT TCC TCA CCG CCA TGT TCA CCC TCA CCA TGT GGG TGA TGC

TGC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F092
TAT ACG CAG TGC TAC CAT CGA TCA CAA ACC TCC AGA AGG AAC TCA TAG TTC CTT
582

CCA GGA GTT TGA TTT TGA TGA CCC AAT CCC CAC GTG CTT GGA AGT TCT TGA AAT

CTG TCC ACC TTC CCA TTT ACT GCA GTT GGG AGC TGT GTG ATT TGG GCA TGT GGC

AGA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F093
TAT ACG CAG TGC TAC CAT CGA TCA CAA CTT CTG CCT TTG TTG GCT GTA GGC TTG
583

ATG ATT CGC CCC AAG ACT GAG AAC CTT GAT GTC ATT GTC AAT GTC AGT GAC ACT

GAA AGC TGG GAC CAG CAT GTT CAG AAG CTC AAC AAG TTC TTG GAG CGT GAG TGT

GGG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F094
TAT ACG CAG TGC TAC CAT CGA TCA CCT GGT TAT GTG TCA GTT CAA GAC TTC GGG
584

CAG GGG ACT GGG GAC CTT GGA AGT GGA ACA TCT GGC CCC TGA GTC TCT CCC

TCC CAC CTC TTT AGC TTA CAA CGA CTC TAT CCA AGC CCA AAA GAA TGA TGT CTG

CCG CCC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F095
TAT ACG CAG TGC TAC CAT CGA TCA CGG ACG CTA TTA CGA ACA GCC AGA TAA TGG
585

AGT CCT CAA CTA CCC CAA ACG TGC CTG CCA ATT CAA CCG GAC CCA GCT GGG CAA

CTG CTC CGG CAT TGG GGA CTC CAC CCA CTA TGG TTA CAG CAC TGG GCA GCC

CTG TGT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F096
TAT ACG CAG TGC TAC CAT CGA TCA CTT CAT CAA GAT GAA CCG GGT ATC TAT GAC
586

CTT GGT CCC CAG GGT GAA TGG AGG AAG GAT CTG GGG ACA CCA CCT GCA GAC

AAT TGC ATC CTT TCA CTG GGG CTA ATG GGC ATG AGA AAG ACT TGG ATG TTT GTG

TAG CTG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F097
TAT ACG CAG TGC TAC CAT CGA TCA CTC CCC GGC TTA GCT TGG TCT GGA TGC CCA
587

TCT TCG ACA ACT TCT TCC TCT GAC TCT CTT CAC CTT CCA CCC TCA CTC CAG GTC

ATC AAC TTC TAT GCA GGA GCA AAC CAG AGC ATG AAT GTT ACC TGT GCT GGG AAG

GTG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F098
TAT ACG CAG TGC TAC CAT CGA TCA CCG CAC TCC TCT TGC TTC TCT CTG GGA TGC
588

AGA GGC CTG CTC TCC TAG GGG CCA GAC ACA CGC CCT CCT CCA CCA ACG CCC

TGG CCT CTG GCT TCT CTC CCT AAC GCT TCC ACC TTC TCC TTC ATT CCC AGA TTG

TCC GTA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F099
TAT ACG CAG TGC TAC CAT CGA TCA CCC GGT CTG TCC TTT CTA GAA ACT GGC TGC
589

TCC CTC CAC ATC CCC TTC CTT GCT TCC TAT TCA ACC CTT AAT CAT GTA TCT CTT

CTT TCT TGG CTC TGC TCC AGA AAC TGA TTC CTG AGG ATG GGG TAA GAA CTT GGG

GTA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F100
TAT ACG CAG TGC TAC CAT CGA TCA CAC AGC GAG ATG AAG ATG CTG AGA ATC TCG
590

GCA ACT TCG TCA TGT TCC CCG CCA ACG GCA ACA TCG ACC TCA TGT ACT TCC CCT

ACT ATG GCA AAA AGT TCC ACG TAA GTC CCA GGG GAG GCC CAG GCT GAT GGC

GGG TGC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F101
TAT ACG CAG TGC TAC CAT CGA TCA CAA CGT GGA GGT GAA TGT AGA ATG TCG CAT
591

CAA CGC CGC CAA CAT CGC CAC AGA CGA TGA GCG AGA CAA GTT CGC CGG CCG

CGT GGC CTT CAA ACT CCG CAT CAA CAA AAC CTG AGG CCC CTT CCT CCC ACC CCA

TCT CTC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F102
TAT ACG CAG TGC TAC CAT CGA TCA CCC TGT GGA TGC TCC TGG AAT GTC CCT GAC
592

CCT GCC TGA TCC CTC CCT CAC CCA CCC CAA AGG TAT TTT TGA TAA CAG AGC TAT

GAC TTG TCT GAG CCT CAC ATC CTT TTC CTT GAC TTC TCA ACC CAG CCT GAA GTC

CAT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F103
TAT ACG CAG TGC TAC CAT CGA TCA CGC GGT TCC GTC ACT CGC CTT TCC CAC CAA
593

CTT CTC CCA ACC TCA GAT CAG TCA GAC AGG GAG CTG GGC TAA GAT GGC CAC

GGA GGA GTT AGG AGC CTT TCT AGT TCT GGT TTA GCT GTG AGA GCT ATC CAC TCT

CCT GCC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F104
TAT ACG CAG TGC TAC CAT CGA TCA CAA CGT GCA CAC GCG TCT CAT TTG ACC CCT
594

TTG CTT CCA GAG ATG AAT GTG GCA CTC CCT CCT TCC ATT CCT AAG CTC TGG CCA

CCG TCC CTT GAT CTC TCA TAC TTT CTC CCT GTC TAC ACA GTC GCC ATC TTG GTG

ACT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F105
TAT ACG CAG TGC TAC CAT CGA TCA CTG AAT TTA TCT GGC TCC TGG GCA GGT CTT
595

CTC CTC CTC TCC ATC CCT ATT CCC TCC TCT GAA ATG CAC CCC TTT GTA ATT GAG

GAC AAG GTG GTT CTG TGG CCT TTT CCC TCT TTG CTG GCA CGT TCT GCT TCT CAC

CCT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F106
TAT ACG CAG TGC TAC CAT CGA TCA CAA CCC ATA ATG CCC ACA GAA TGT CAA ATG
596

AGG GGC CTC CTG CCT CCT GCT CTG AAT ATT CTG TAG CTG TAG AGG CAT TTT AAC

CCT TTG TCC TCC AGC ATC CCT TCA CTT CCT CAT CCT CTC TAA CCT CCT TTT TCT

TTT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F107
TAT ACG CAG TGC TAC CAT CGA TCA CGG AAG CTC ATC AAG GTG ACC TGT CTA CAG
597

AGG CAA GGA CAG GGA CTG AGC TTC AGG AGC TCT AGT TTG CCT GCT GGG TAG

GGA CAG ATG TTT AAG TTA AAA GTC TCT GAA AGA GGC GGG TCT GGA TCT CCT GGG

GAG AGT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F108
TAT ACG CAG TGC TAC CAT CGA TCA CTT TGG CAT TCC CTA GTA AGA AAG AGA AAA
598

AAA AAA GGT GGT CTT GAG GTC AGG AAG GCT GGT GGC TTC ATA GCT GTC TGG GAT

CCT TGG GGC AAG GCA AGG GGC TCC TGC TTG CAC CTT CAG CCT GGT TGA TGA

GCT GAC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F109
TAT ACG CAG TGC TAC CAT CGA TCA CCT CCC TGG GAG CCA GCA GCC CTG AGG
599

AGC ATG GGC AGG CAG TAC TGA GCT CCT CAA CCC GAC TCT CCT CCC TAT CCC AAG

AAG CCC TTT GAA AGG TTT TCC TGG CAG AGT TTA AAG CTT CAA TTC ATT CAG CTA

CCT GGC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F110
TAT ACG CAG TGC TAC CAT CGA TCA CGA CCC TGG CTA GCA GGC CTC CCA CTG
600

GCC TCT CTC CAT CCA GTA GCA CCC ACC CCT GTT CCC CTT GGG AAC CCA GGT ATC

CTG CCA CTT TCT GAT GGA GCA GAT GGC CAC CCT GGA GGC TCA GCC TTG CTA AAT

CAG ACA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F111
TAT ACG CAG TGC TAC CAT CGA TCA CCA GCC CAC ACT CAT TGC AGA CTC AGG TGG
601

CTG CTT CCC AGC ACC TCC TCA CTC ACC CCT GCA CCT GCT GAC CCC AGT AGC CTG

CAC TGG CGT TCA CCC CTC AGA CAC ACA GGT GGC AGC AAA GTT TTA TTG TAA AAT

AAG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F112
TAT ACG CAG TGC TAC CAT CGA TCA CAA TTG ACC CTG AGC ATA AAA CAA GTC TTG
602

GTG GAT CCA GAT CAT CAT ATA CAA GAG ATG AAA TCC TCC AGG GTG TGG GAT GGG

GTG AGA TTT CCT TTT AGG TAC TAA GGT TCA CCA AGA GGT TGT CAG ACA GGG TTT

GGC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F113
TAT ACG CAG TGC TAC CAT CGA TCA CGA AGT GGG CCC CTA CCT AGA ATG TGG CTG
603

ATT GTA AAC TAA CCC TTA ACT GCA AGA ACA TTT CTT ACA TCT CCC AAA CAT CCC

TCA CAG TAA AAA CCT TAA AAT CTA AGC TGG TAT GTC CTA CTC CCC ATC CTC CTC

CCC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F114
TAT ACG CAG TGC TAC CAT CGA TCA CCA ACA AAA CAC CAG TGC AGG CCA ACT TGT
604

TCA GTG GAG CCC CGG GAC AAA GCA AAT GGA AGT CCT GGG TGC TTC TGA CGC

ACA CCT ATT GCA AGC AAG GGT TCA AAG ACC CAA AAC CCA AAA TGG CAG GGG

AGG GAG AGA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F115
TAT ACG CAG TGC TAC CAT CGA TCA CTG GGA GGC TGT CAG TGG GGA ACA AGA
605

AGT GGA GAA TGT CAG TCT GAG TCA GGC CCT TCT GTC TTG AAC ATG AGT TTT TTA

TGG CGG GAG GTA GAC TGA CCC TTT TTG GAC TTC AGG TGG CTG TAG GAG ACA

GAA GCA GGG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F116
TAT ACG CAG TGC TAC CAT CGA TCA CGG AGA GAT GAC ATC ACA TGA GTG AGA GGG
606

TCT GTG CCC CTT TTC CCT GAC CAA TGC TTT GAA GGG CCT AAG GCT GGG ACA ACG

GGA ATT CAA ATC AAG ATG GTG GCC ACA CCC CAT GCA AAT ATG TTT ACT GAG CAC

CTC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F117
TAT ACG CAG TGC TAC CAT CGA TCA CGG TGC ATG GCA GGG CTG AGT ATA TGA CCT
607

GAA ACT CTG GCT GTA TTC AGT ATT ACA CAA TTA TTA GGC CCC TCC TTG AGA CCC

TCC AGC TCT GGG CTG GGA GTT GCG GAG AAT GGC AAA GAA GTA TCC ACA CTC

GTC CCT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F118
TAT ACG CAG TGC TAC CAT CGA TCA CGG TTT GGA TGT TCT GTG GAT ACA CTG AGG
608

CAA GAA TGT GGT TAT AGG ATT CAA CCG GAG GAA GAC TAA AAA AAT GTC TGT GCA

GGG CTG GGA CCC AAT GAG ATG GGG TCA GCT GCC TTT GAC CAT GAA GGC AGG

ATG AGA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F119
TAT ACG CAG TGC TAC CAT CGA TCA CCA GTC AAG AAG AAA ACG GCA TTT TGA GTG
609

TTA GAC TGG AAA CTT TCC ACT TGA TAA GAG GTC CCA AGA CTT AGT ACC TGA AGG

GTG AAA TAT TCT CCA TCC AGT GGT TTC TTC TTT GGC TGG GGA GAG GAG CTG GTG

TTG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F120
TAT ACG CAG TGC TAC CAT CGA TCA CCA GCT CGT GGT GAG GCT CCC CTT TCT TGC
610

GGA GAT TCT CTT CCT CTG TGC GCC GGT CTC TCC CAG GAC AGG CAC AAA CAC

GCA CCT CAA AGC TGT TCC GTC CCA GTA GAT TAC CAC TAC TCA GGA TAG GAA AAG

AGA AGC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F121
TAT ACG CAG TGC TAC CAT CGA TCA CAG AGG CAG TAA GGA AAT CAG GTC CTA CCT
611

GTC CCA TTT AAA AAA CCA GGC TCC ATC TAC TCC CAA CCA CCC TTG TCC TTT CTG

GAG CCT AAG CTC CAG CTC CAG GTA GGT GGA GGA GAA GCC ACA GGT TAA GAG

GTC CCA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F122
TAT ACG CAG TGC TAC CAT CGA TCA CAG CCA GAG AAA AGA AAA CTG AGT GGG AGC
612

AGT AAG GAG ATT CCC CGC CGG GGA TGT GAT GAG AGG TGG ATG GGT AGT AGT

ATG GAA GAA ATC GGT AAG AGG TGG GCC CAG GGG TCA GAG GCA AGC AGA GGC

TGG GGC ACA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F123
TAT ACG CAG TGC TAC CAT CGA TCA CCA GGC CAG TGT GCA GGG TGG CAA GTG
613

GCT CCT GAC CTG GAG TCT TCC AGT GTG ATG ATG GTG AGG ATG GGC CTC CGG

TTC ATG CCG CCC ATG CAG GAA CTG TTA CAC ATG TAG TTG TAG TGG ATG GTG GTA

CAG TCA GAG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F124
TAT ACG CAG TGC TAC CAT CGA TCA CCT ACT GCT CAC CTG GAG GGC CAC TGA CAA
614

CCA CCC TTA ACC CCT CCT CCC AGA GAC CCC AGT TGC AAA CCA GAC CTC AGG

CGG CTC ATA GGG CAC CAC CAC ACT ATG TCG AAA AGT GTT TCT GTC ATC CAA ATA

CTC CAC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F125
TAT ACG CAG TGC TAC CAT CGA TCA CCG CAA ATT TCC TTC CAC TCG GAT AAG ATG
615

CTG AGG AGG GGC CAG ACC TAA GAG CAA TCA GTG AGG AAT CAG AGG CCT GGG

GAC CCT GGG CAA CCA GCC CTG TCG TCT CTC CAG CCC CAG CTG CTC ACC ATC

GCT ATC TGA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F126
TAT ACG CAG TGC TAC CAT CGA TCA CTG TGG AAT CAA CCC ACA GCT GCA CAG GGC
616

AGG TCT TGG CCA GTT GGC AAA ACA TCT TGT TGA GGG CAG GGG AGT ACT GTA

GGA AGA GGA AGG AGA CAG AGT TGA AAG TCA GGG CAC AAG TGA ACA GAT AAA

GCA ACT GGA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F127
TAT ACG CAG TGC TAC CAT CGA TCA CAT ACG GCC AGG CAT TGA AGT CTC ATG GAA
617

GCC AGC CCC TCA GGG CAA CTG ACC GTG CAA GTC ACA GAC TTG GCT GTC CCA

GAA TGC AAG AAG CCC AGA CGG AAA CCG TAG CTG CCC TGG TAG GTT TTC TGG

GAA GGG ACA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F128
TAT ACG CAG TGC TAC CAT CGA TCA CAC CCT TCC CCA CCT GAT ACA CGG CTC CAT
618

TTC TTT GAT TCC TTT CAC TGC AAA GCT TCT GGA AGA ACA ACT GTC TCA CCG CTC

ACC TGC CCA TTC TCT TCG GAC ACT CCT CAG CCC TGC ATT ACA AAC CCC TCA CGA

ATG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F129
TAT ACG CAG TGC TAC CAT CGA TCA CGA ATA ACA CAC AAG CCT GTT ATA TGA GAG
619

GTT AAG AGA GCG AGA AAG AGC AAG GGG CAG CCC CTG TGT GGA CCA GCA TCT

TGC ACG AAG TTA TGC AAC TAT CAT CGC ACC TTC TCC CAG ACA AGC TTT CAA AGG

CTT TGC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F130
TAT ACG CAG TGC TAC CAT CGA TCA CGC CCA AAG TCT CAA TCC CAC TTG GAG GGA
620

CAC AGG TCT ACA GAC AGG TCT CCC TGT CTT TAT CTC TCA AAT CTT CAG TAG CAA

CTA AAA TCT CCG TGT TTT TCA GAG CAG GAC CTT CCC AGG GGT ACC AGC ATC AGT

GGG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F131
TAT ACG CAG TGC TAC CAT CGA TCA CCA GGA TAC AAA TGT GCC AGG CTG AAC TAG
621

GCC TTC CAA ATG GCC AGG GAG CCA AGA GAA ATG CAG GTG CCC TTG GCT GGG

TGG GAA GGC AAT GAG ATC AAC TGA GAC CCC AAA CAG GGG CAG GCC TGA CCA

GAA TCT TAA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F132
TAT ACG CAG TGC TAC CAT CGA TCA CAG ATG ACG TAA GTA CGG CAC AAA GTG GCC
622

GGT ACG CGG CAG GTG CAT GGG AAG AAA CTG CGG AAT GAA ACA ACC GCG AGC

TAA GAG ATG GGG CAG CGG GAG AAA TGA ATT CGA GTT CCG CCT CCT ACC AGG

AAG AAC CGG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F133
TAT ACG CAG TGC TAC CAT CGA TCA CTC GGG CCG GAG GGC TGC ACG GAG GAC
623

CAC ACG GAC GCC TGC GGG CCC GCC CCT TCC GCT TCA CGA CGT TCA GCC TGC

GTC TGG AAC TGG AAT GGC CTA GCC CAA AGC TAG ATA ACA GGT AGA TTG TTT TTC

CGA CAA ATT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F134
TAT ACG CAG TGC TAC CAT CGA TCA CTT CAA AAT TTG ATT CTC AGA CGT ACC CAT
624

TCT TTT TTT TTT TCC TCC GGG AAG ATG AGA TAT ACT CAT TCT TGA AAA TAC CTC

CGG GCT TGC CTT CTG CAC ACT TCT TTC CCT CCC TGT CTC ACG CCA TGG TAG CGT

CCG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F135
TAT ACG CAG TGC TAC CAT CGA TCA CCT AGG TTG CAG GCG ACC CGC GGG GTG
625

GGG CAC ACC ATT CAA AGA AGG GGA GGG ATT GAG GTT TGC ATC AAA ACA AAT ACC

CCT GCC TTT GCA AAG GCC ATA ACT AAG TAA TCC AGA AAA AGA AAT GCA GGC GGA

GAA TAG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F136
TAT ACG CAG TGC TAC CAT CGA TCA CAG CCT CCC TCT GCC AAG TAA GAG GAA CCG
626

GCC TAA AGG ACA TTT TCT CTC TCT CTC CTC CCC TCT CAT CGG GTG AAT AGT GAG

CTG CTC CGG CAA AAA GAA ACC GGA AAT GCT GCT GCA AGA GGC AGA AAT GTA AAT

GTG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F137
TAT ACG CAG TGC TAC CAT CGA TCA CTC GGA ATG GAG CCC CAG TTT TCA CTA GGA
627

TGC CAT GGG CTC TAA AAT ATA CAG CTA TGA GTT CTC AAT GTT TCG AGA TCC AAA

AGT CTC AGA CCT CAA TGC TTT GTG CAT CTT TTA TTT CAG GGA TTC CCT ACG CCC

AGC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F138
TAT ACG CAG TGC TAC CAT CGA TCA CCC GGG TGG ATG TGC AAA GAA GTA CGC TTT
628

AGG CCG GCT CAA GGT TCC CCA AAG CTC CAC TCC TCT GCC TAG GCG TTC AAC TTT

GAG TTC GGA TGG TCC TAA CAT CCC CAT CAT CTA CAC CCA GGT CTC CCA ACA ATG

CAA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F139
TAT ACG CAG TGC TAC CAT CGA TCA CAG CCC CAG CGA TTT TCC CGA GCT GAA AAT
629

ACA CGG AGC CGA GAG CCC GTG ACT CAG AGA GGA CTC ATC AAG TTC AGT CAG

GAG CTT ACC CAA TCC AGG GAA GCG TGT CAC CGT CGT GGA AAG CAC GCT CCC

AGC CCG AAC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F140
TAT ACG CAG TGC TAC CAT CGA TCA CCA AAG TGT CCC CGG AGC CCA GCA GCT ACC
630

TGC TCC CTG GAC GGT GGC TCT AGA CTT TTG AGA AGC TCA AAA CTT TTA GCG CCA

GTC TTG AGC ACA TGG GAG GGG AAA ACC CCA ATC CCA TCA ACC CCT GCG AGG

CTC CTG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F141
TAT ACG CAG TGC TAC CAT CGA TCA CCA CAA AGC TGG ACA GTC GCC ATG ACA AGT
631

AAG GGC AAG TAA TCC GCC TGC CGG AGG AAG CAA AGG AAA TGG AGT TGG GGA

GGA GGG TGC AGA GTC AGG ATT CTC GCC GAC CTG GTG CCG TAG ATA CTA ACA TTT

TGG GGT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F142
TAT ACG CAG TGC TAC CAT CGA TCA CGT AGG CGC TTC TCG CCA AGA TAG AAG CGT
632

TCA GAC TAC AAC TCC CAG CAG CCA CGA GGA GCC CTA GGG CTT GAT GGG AAC

GGG AAA CCT TCT AAC CTT TCA CGT CCC GGC TCC GCG GGT TCC GTG GGT CGC

CCG CGA AAT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F143
TAT ACG CAG TGC TAC CAT CGA TCA CGC CCA ATC GGA AGG TGG ACC GAA ATC
633

CCG CGA CAG CAA GAG GCC CGT AGC GAC CCG CGG TGC TAA GGA ACA CAG TGC

TTT CAA AAG AAT TGG CGT CCG CTG TTC GCC TCT CCT CCC GGG AGT CTT CTG CCT

ACT CCC AGA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F144
TAT ACG CAG TGC TAC CAT CGA TCA CGA GGA GGG AAG CAC AGG TGG GTT TCT TTA
634

GCT CTG CGT CGG ATC CCT GAG AAC TTC GAA GCC ATC CTG GCT GAG GCT AAT CTC

CGC TGT GCT TCC TCT GCA GTA TGA AGA CTT TGG AGA CTC AAC CGT TAG CTC CGG

ACT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F145
TAT ACG CAG TGC TAC CAT CGA TCA CAC CCA GTT TCT CTC TCC ACT CCC CTG GAA
635

ACA GAG TTT GGT TCC CCT AGT GAG TTG AGT CCT CGA ATC GAG GAG CAA GAA CTT

TCT GAA AAT ACA AGC CTT CCT GCA GAA GAA GCA AAC GGG AGC CTT TCT GAA GAA

GAA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F146
TAT ACG CAG TGC TAC CAT CGA TCA CCG AAC GGG CCA GAG TTG GGG TCT GGA
636

AAA GCC ATG GAA GAT ACC TCT GGG GAA CCC GCT GCA GAG GAC GAG GGA GAC

ACG TAA GTG GTG ATG GCA GTG GAG TGT GGA GTC TGG GGA GAT GAA GTG TGA

GGT CGA TCT GTC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F147
TAT ACG CAG TGC TAC CAT CGA TCA CAT CTT CCT TTC AGC GCT TGG AAC TAC AGC
637

TTC TCC CAG CTG CCT CGA TTT CTC AGT GGT TCC TGG TCA GAG TTC AGC ACC CAA

CCT GAG AAC TTC TTG AAA GGC TGT AAG TGG TAA GGA TAA CAA CGG GGC AGG

GAG CTG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F148
TAT ACG CAG TGC TAC CAT CGA TCA CGC TTA CCT ACC CCA GAG GCA GGC TCA GCC
638

CTA GCC CTA CAC TTG AAA AGC ATA GGT CTG GCC AGC TTT CTA ACT CTC CCC TGT

TTC TAG GGC TCC TGA CGG TTC CTG CAT CTT GAC CAA TAG TGC TGA TAA CAT CTT

GCG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F149
TAT ACG CAG TGC TAC CAT CGA TCA CAG CAG GTG GAA TAT GCA GAA ATG GTA AGG
639

ACT GGG GCT AAC TGC CTC TTC ATC AAT GCT GCA CAT TTA AGT CCT TCG TGG AGA

TGG AAA AAG TGT AGT CCA AGT GTT TCC TGT TCA CAA ACG GGA CTG TTT TTC AAG

ACG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F150
TAT ACG CAG TGC TAC CAT CGA TCA CTT CCA TAT ACT TGT GTG TAC TGA GTC TTC
640

TTC CCA ATA TAG TTT CTC GGG TTT TCT CCT TTC TTT TCC ACC TTT TCA CAG AAC

TTT CTG CAA GTC TCA TTT CCA GCT CCC CAG CGT CTT TCC TGA GTA CTT GCC CTG

CCC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F151
TAT ACG CAG TGC TAC CAT CGA TCA CGT GGG TCT TGC AGG AAG CCT TTA CTC CTC
641

GTC TTG CCC TCT CCA GGA GAC ACT TTG CAT CCT CTG TAC CCT TTA TCT CTC AGG

GTG GGG ACG GGG AAT GTC CTC ATT CCC AAA TGC TGT AGC CAC ACA ATT GCT CTT

TCG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F152
TAT ACG CAG TGC TAC CAT CGA TCA CCC TTT TGC TCA TTA AAA CCG TAT TTG TTG
642

ACT CTG CTT ATT CTG CAC CAC GTG TTG GGG AAG CAG TGG TGA GAG CGA GGC

AGA TGT GAT TCC CTC CTT CTC TGA TAG GTA TGA CGG AAG GGG AGT GAG GAG CAC

CAG GGG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F153
TAT ACG CAG TGC TAC CAT CGA TCA CAA AGT ATG AAT CTA TAT GGC TTT TGG TGG
643

CTA AAT TTG ACA TTA AAG TCT GAG CTC ACC CTT GAA CAT TGA GAC AGA GTC TGT

GCT CCA TAT ATA CAC CCC ATC TGC CAC AAC ACT GCT AGA GGC ACG CGC CTC AGA

CTC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F154
TAT ACG CAG TGC TAC CAT CGA TCA CGT CTC TGT ATA GGT CCC TGT CCT TCG AAT
644

GGT GGA AGG TGA TAC CAT CTA TGA TTA CTG CTG GTA TTC TCT GAT GTC CTC AGC

CCA GCC AGA CAC CTC CTA GTA AGT AAT GTT TGC CTC CCT GCT CGC CGC CCC ACC

ACC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F155
TAT ACG CAG TGC TAC CAT CGA TCA CAT TCC TCC CCT TCC TTT GAC AGC ACC GGG
645

GTT TCA GTG TCC ATG TCT CTC TCA GCG TGG CCA GCA GCA GCC GGG AGA ACC

CGA TTC ATA TCT GGG ACG CAT TCA CTG GAG AGC TCC GGG CTT CCT TTC GCG CCT

ACA ACC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F156
TAT ACG CAG TGC TAC CAT CGA TCA CCA TAC CCT GTC AGC TGT GGA GCT TTT GGT
646

CTC TGA AAT CTT TCT AGA AAA TTG TTG ATA AAG CTG ATT CCG TTT TCC TGT AGG

CCT TCA ACT TGC ATC TCT CCA AGG AAG AAC TGG GAT TTG AGA GGG ATG AAG TGG

GGC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F157
TAT ACG CAG TGC TAC CAT CGA TCA CAC TTT GTT CCT TCC CTC TCT AGC AAA AAA
647

GCA GGG CCA GAG CGG CAT CAT CTC CTG CAT AGC CTT CAG CCC AGC CCA GCC

CCT CTA TGC CTG TGG CTC CTA CGG CCG CTC CCT GGG TCT GTA TGC CTG GGA

TGA TGG CTC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F158
TAT ACG CAG TGC TAC CAT CGA TCA CTT TCA TCC CGA TGG CAA CCG CTT CTT CTC
648

AGG AGC CCG CAA GGT AGG GGT CAC ACC CTG AGA GCC CAA AGC AGC TGG GCA

GCG GGG CAG GAG CAG GGA TGT AGT CTG CAG TGT AGG GGA ATG GGT GGG GAT

GGG GAA AAA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F159
TAT ACG CAG TGC TAC CAT CGA TCA CTG ACT CCA GGT CCT GTT CCT TGT CTC CAG
649

GAT GCT GAG CTC CTG TGC TGG GAT CTC CGG CAG TCT GGT TAC CCA CTG TGG

TCC CTG GGT CGA GAG GTG ACC ACC AAT CAG CGC ATC TAC TTC GAT CTG GAC

CCG TGA GTG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F160
TAT ACG CAG TGC TAC CAT CGA TCA CAG TTC CTA GTG AGT GGC AGC ACG AGC
650

GGG GCT GTC TCT GTG TGG GAC ACG GAC GGG CCT GGC AAT GAT GGG AAG CCG

GAG CCC GTG TTG AGT TTT CTG CCC CAG AAG GAC TGC ACC AAT GGC GTG AGG

TCC TCA GTT CAA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F161
TAT ACG CAG TGC TAC CAT CGA TCA CTC AGG GTG AGC GGG GCT GAG CAG GAG
651

CTG GGT CAG ACT GTT GGG TGT GAC CGT GTG AGA CTG TGC AGA CAG TGA CAG

GGC GCC TGT CGC CCA CAC TCC ACA CTG TTT GCC ATT CTC CAG CAG CAT GGG

GAC CAT TAC TCA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F162
TAT ACG CAG TGC TAC CAT CGA TCA CCT CTG AGG GGA GCA GGA GCA CTC TCC TCC
652

GCT CCC CGG GGC TCC CCA GGA GGC AGA CAA CCC AGT TGC CAG AGT CAA GGA

CAC ACA TAA AAG AGT AAA GGT GTT GAA AAA TAC AGT CGT CAC CTT TGA TAT TGC

TGT TCC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F163
TAT ACG CAG TGC TAC CAT CGA TCA CGA CTT TGA AGA AAT ACT GCC AGC AGG GGA
653

TGT GAT TTT GGA CCT CAC AAT CAA AGG GAA GGG ACG AAA GGC CTG GGG AGG

CTT GAG AGG GAG AGG AAT GTC ACC CCT GCC CAG AGG TGC TGA AAG CCA GGG

CTC TTA CAC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F164
TAT ACG CAG TGC TAC CAT CGA TCA CCT TCA AGG TTA TTA TTA TTC TCT CCA AAC
654

CTG CCG GGA GCA GCG GTG TTG TTT TGG GAT GGA GGA GGA GGC TGC GGG AGC

GAA AGG GGT GGG TTC CTC GGG GTG GAG AGG GCG AGA GCC TTT CTG GAT TCG

AGA GAG GAA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F165
TAT ACG CAG TGC TAC CAT CGA TCA CCT TCT GTG GGC AGG GAG GAG GCG GGA
655

GGG AAG GTG CTG GTG CTC TGA TGT GTG ATG GGT TAC TAG ACA GGT GAT CTT

GGG AGC CAG ACT CCG GGT CCC ACG CAG AGC TGG ATG CGG GTG GTG CTA TGG

ATG TCA GGA GTT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F166
TAT ACG CAG TGC TAC CAT CGA TCA CCT CTG GGG ACA ACT TCC CCA AGG CTC CTT
656

GAC TCT CTT CAC AGT CTG TCT CCC AAC TCT TCC CCA CAG CCA GAG GGT CAG TGA

ACT GTT GGC TGA ACG ATT TCC CAG CCA GAA CAT ATT CCT GCC TTC CTG GCT TGC

CGG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F167
TAT ACG CAG TGC TAC CAT CGA TCA CAA TCT TTG CTG CAA ACT CTT CCA TCA GAC
657

CCT CTG TTC CAT GGC ATG CCA GCT TTG CTC TGA AAA GAG GCC AGG GGT CAG ATG

AAA CTT CAA GCC ACG CTG GGT AGG ACA GAA CCT TCG GGA GGT CAC CTG GGT

CCC TTG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F168
TAT ACG CAG TGC TAC CAT CGA TCA CGC GGT AAG GTG TTT GAA TGT ATT ATG TGC
658

TCA TTA AAG GAG AGC TAG GAT TAT TGT TCC TCT CTT TAC TTC CCA GCC TCC CTC

ACA CTT CTC TGC TCT GCC ATC CCT CCC TCT CTT TTC CCT GGA TCT CTT TGG GTA

TAA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F169
TAT ACG CAG TGC TAC CAT CGA TCA CCT CTT TCG GAT GTG GGG TGG GAG TGG
659

GAC ACT TGG GAG TCT GGG AAG ATA TCA AGT AGC AGT CCC CTG GGA CCC AGT

CCT GAG ACC CTG TCT CAG CAG CTA TTG ATG TCC AGG AAG GGG CTG CAG GGG

TTG GCA AGT TGT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F170
TAT ACG CAG TGC TAC CAT CGA TCA CGC TGT GCT GGG ACA GTG GAT GCC TGG
660

GTG CAC TGG CCA AGG AGA TGG TGA AGT GTG TTG GTT GTG GTT AGA AAA GTC AAC

TCC TTT CCT TTC CAA AGC AAT AGA GCA CTT GCC CCA GAA GTC TAA GAC CCA GTG

TGG GAT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F171
TAT ACG CAG TGC TAC CAT CGA TCA CCT GAG TGG GAG AAT GAC TCA GGC AGC
661

AGG TTC CTA GAC CCT GGT TCC CAT CAG CCC CAA TGA TGG TCG TGG CCA AAC CAG

GCA TTT GCC TTC TGT GCT ATT AGC TGG CTA ACT TAG GAC ACT GGT CTG GAC CAC

CCT CCA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F172
TAT ACG CAG TGC TAC CAT CGA TCA CTT TGC CCG GAA ATC CCT GAA ATT CAG TGG
662

TGG CCT GAA GGA GGG GAG GCT CTG CCC GCA TGG TTG GCT GCC ATG GAA TAG

TGA AAT CAC CTG GGA GGG GTG GGC TGT GTG GTT CCA GAG AGG CCA GCT CCT

TGG TAA CTG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F173
TAT ACG CAG TGC TAC CAT CGA TCA CTG GTG ATG GAG CGG GAG ATG GCG GTG
663

TGC ATG TGG TGA GGG CGG GCT GAA GAG TGG AGT GCA TTT GGG CAC ACC AAG

GGG CAG GAG ACC CCT GAG CCT GGC TTC CTG CTG CTT CCA ATG TGA ATG CAC

AGA GTC CTT GGT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F174
TAT ACG CAG TGC TAC CAT CGA TCA CTG CCC CAA ACC TCC TTC TCA CTT GTG ATC
664

GCC CAG ACC TGG ATC TCC GCT TCA CCA TCA AGT TCC AGG AGT ATA GCC CTA ATC

TCT GGG GCC ACG AGT TCC GCT CGC ACC ACG ATT ACT ACA TCA TTG GTA CTG CTG

GGC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F175
TAT ACG CAG TGC TAC CAT CGA TCA CTG GGG CCA GAA TCA GGG CTA GAT TCT GGA
665

GTG CCA ACC TCT TCC TCT GGC TTT TCT CTC CCA GCC ACA TCG GAT GGG ACC CGG

GAG GGC CTG GAG AGC CTG CAG GGA GGT GTG TGC CTA ACC AGA GGC ATG AAG

GTG CTT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F176
TAT ACG CAG TGC TAC CAT CGA TCA CCG GTG ATA CAG GAA AGA GGA GAA GAG
666

AGG ATG GGA GGG TGG GAG GGG AAT GGA AAC CAA ATG AGG AAA AGA CTC AAT

TAG AAC TAA TTA GCC AAG TCA GTG CTT CAA TCA GTG CTG TCA GAG AAG TGG GGA

GGA CTC CGT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F177
TAT ACG CAG TGC TAC CAT CGA TCA CTC ACC CAC CCA GAG CTA GGG GCG GGA
667

ACA GCC CAC CTT TTG GTT GGC ACC GCC TTC TTT CTG CCT CTC ACT GGT TTT CTC

TTC TCT ATC TCT TAT TCT TTC CCT CTC TTC CGT CTC TAG GTC TGT TCT TCT TCC

CTA GCA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F178
TAT ACG CAG TGC TAC CAT CGA TCA CCC TTA TGG GGA AGG CTC TGA CAC TCC ACC
668

CCA GCT CAG GCC ATG GGC AGC AGG GCT CCA TTC TCT GGC CTG GCC CAG GCC

TCT ACA TAC TTA CTC CAG CCA TTT GGG GTG GTT GGG TCA TGA CAG CTA CCA TGA

GAA GAA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F179
TAT ACG CAG TGC TAC CAT CGA TCA CTG TCC CGT TTT GTC CAG TGG CCA ATA GCA
669

AGA TAT GAA CCG GTC GGG ACA TGT ATG GAC TTG GTC TGA TGC TGA ATG GGC CAC

TTG GGA CCG GAA GTG ACT TGC TCC AGA CAA GAG GTG ACC AGG CCC GGA CAG

AAA TGG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F180
TAT ACG CAG TGC TAC CAT CGA TCA CAT CAG GAG GTG GGA GGT GGA TGG TTC TTA
670

TTC TGT GGA GAA GAA GGG CGG GAA GAA CTT CCT TTC AGG AGG AAG CTG GAA

CTT ACT GAC TGT AAG AGG TTA GAG GTG GAC CGA GAA GGA CTT TTC CCA GTC TTC

AGT GGC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F181
TAT ACG CAG TGC TAC CAT CGA TCA CTC CCA AGA TCT CCC TTC CCT TGT GCT CTG
671

TGC TGA TTT TAG GAC AGC TAA GAT GAC TGC CAT GTG CTG TGG CAG GCC TAA TTT

GTC TTG TTC TTT CCT TTC CAT ATC CCA GTA TAA TCT CTG TTA ATC AAC AGG ACT

ACC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F182
TAT ACG CAG TGC TAC CAT CGA TCA CCA AGA ACC CAT GTG CTC TCC CGA GTA ACC
672

CAG ATG GCT GTC TTG TTC ATT CCA TCC TAC ATT TCT GAC TCC TTT CAG ACT CAA

CAC AGT TCC CTT CTT AGT GAC CAA AAT GGT GGC CTA CTG GCT GGT CTA GCT GAC

AGT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F183
TAT ACG CAG TGC TAC CAT CGA TCA CGT ACT TAG CAA AGG CCA CTG TTT CCA TAG
673

TGA CCA GCT GAT ACC TCT TCC TGC CCT CTA GTG TGC AAT TGG GTG TTG CCT CAG

TTT CCT CCC AGC TCA GTT TTA TTA GAT CAA AGC TGT TGT TGG GCA CCA GGT TGG

CCA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F184
TAT ACG CAG TGC TAC CAT CGA TCA CCT CAA TCA CCA GCC AAG ATG GTT GCT TTG
674

TCC ACC AGA GGT CAA GTT CAC CTC TCT GGT GCT GTA GTT CCC AGC TCC TTC CTG

ATT TTT CTA ATC GCT CCT TCT GGG GAA CAG GAA GTT GAT ATT GCC ATG GTG GCG

GGG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F185
TAT ACG CAG TGC TAC CAT CGA TCA CGT GGG TAG GGA TAG GGC TAG TTT GGA GTG
675

GTG AGG TTG GCA GTG CTT GGG GAG GCG AAA ATG GGC TGG GCG AGA AGG CAG

AGG AAG GTG TCA CCT CTA GGG GAG GGA GAG AGT AAT GCA GGC AGG AGT GGG

GTC GCC AAT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F186
TAT ACG CAG TGC TAC CAT CGA TCA CCA TCC ATG GGG ACA GTC TGA GTG TCT TGC
676

TAA GCC AAA GCA CCA GAG ACA GGG TAA GAC GCA ATT AGG ATC AAA GTG GTA AAA

ACA TGT TCA CAT GTT AAA GTC TAT TCC CTA CCA TCC CTA CCC AAG GCA CTT GAG

AAG GAC TTC GAA AGA CGC TCC ACG ATA T

p53F187
TAT ACG CAG TGC TAC CAT CGA TCA CAA AAA GCC CAG GTG CCC AGT CCC CTA GAC
677

TTA GCC CCA TTC TCC AAC CAC AAA AGC AGC CGT CAG GAG CAT CAC TGG AGC

GAG TCA GCA GGG GTC ACA ACC TCT GCC CTC CCT GCC CCT CCC CTG ACT TTA

GGT CCC CTC GAC TTC GAA AGA CGC TCC ACG ATA T

p53F188
TAT ACG CAG TGC TAC CAT CGA TCA CAG CAA GAT GCC TGG GTC TTG AGA GGT GCG
678

GGC ACC GCA GGA GGG GAG TTG AAA GCT GGG AGG CCA GGT ACT GGG CTG TCT

GGA ATT TAC TCT TGC CTT CTG AGC CCA CAC TGG AGC TGC CCG AAG TGG GGA

GGC TCG TAT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F189
TAT ACG CAG TGC TAC CAT CGA TCA CTC CAC CTA CAT CTA GAG GCA CAG GTG GAG
679

AGA CAA GAG GGA AGA GAC GTG GGG ATA AAT AGT CTC CGT GAC AGA CAA GCA

CCT CGC CAA AGC AGC CAA GAC ATC ATA ACA TGC TGA GAG ACA GGA AGA CAC

CTG GGG ACA GAC TTC GAA AGA CGC TCC ACG ATA T

p53F190
TAT ACG CAG TGC TAC CAT CGA TCA CTC AGA GCG CTG GCA TCC AGA GAC TCC CCG
680

CAG CCC CGC TCG AAT AGC ACA AGA CCA ACA GCC TTT CAC ATG TGT AGT GAC TTG

GGT GTC TGG AGC TAT TTG AAA ATG CTG GTG ACT TCC ACG CAG AAT AAC ACA GGC

CGT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F191
TAT ACG CAG TGC TAC CAT CGA TCA CTC ACC ACC ACA TGC ATG TTT CGG ATG TGT
681

CCT GTT CTT CCC TAG CCT CGG CCT GGA GGG GAG TAA GTC ACT TAG GGT CTT GGA

TCC ACA TTA AGG AAG TTG ATC TCA CTC AGT CTC CCA TCC CCT TGC CTT TTG TTT

CTT GAC TTC GAA AGA CGC TCC ACG ATA T

p53F192
TAT ACG CAG TGC TAC CAT CGA TCA CCT GTG GCA CAG CTG CTG GCA GAG TCC AGA
682

GCA GGA AGG CAG AGC GGA AGT AAA GGA ACC GAG AGG GCT GGG ACC ACA GAA

GCT AGA GCT GTC GCC CTG CCA TCC CCA AAC CGC ATA GGA TTG AGG CTG TCT

TGG GGC TGA GAC TTC GAA AGA CGC TCC ACG ATA T

Table 3 shows the summary of the probe designs and the downstream/cleanup methods.

TABLE 3

PCR

Amination
Linker

Probe

Kb
Steps
Fractioned?
Fraction
or
Removed?

loci
Method
coverage
(1 or 2)
(Yes/No)
Size(bp)
Allylamine
(Yes/No)

HER2
HER2 1-step PCR
76
1
Y
100-300
Amination
NA

HER2
HER2 2-step PCR
76
2
Y
120-180
Amination
N

HER2
HER2 2-step
76
2
Y
100-300
Allyl
Y

PCR-Aminoallyl

dUTP

p53
p53 1-step PCR
60
1
N
50-400
Amination
NA

p53
p53 2-step PCR
60
2
Y
70-300
Amination
N

p53
p532 2-step
60
2
Y
70-300
Allyl (2%)
N

PCRAminoallyl

dUTP

p53
p532 2-step
60
2
Y
70-300
Allyl (3%)
Y

PCRAminoallyl

dUTP

HER2
HER2 oligo
21
NA
NA
NA
Amination
NA

HER2
HER2 Oligo +
76
1
NA
NA
Amination
N

PCR hybrid

p53
p53 Oligo + PCR
60
1
NA
NA
Amination
Y

hybrid

Testing Results

Probes were first tested on male lymphocyte slides, and probe intensity, specificity, background and cross-hybridization were evaluated, with corresponding BAC probe as the control.

All probes tested passed the cross-hybridization test on lymphocyte slides. Once a probe design passed on lymphocyte slides, it was then tested on FFPE slides. All probes were tested on breast cancer tissue FFPE slides. Some probes were tested on both breast and gastric slides. Universal pretreatment method was used for all FFPE slides. HER2 and p53 BAC probes from AM inventory were used as controls for all experiments. BAC control probes passed overnight hybridization on both lymphocyte slides and FFPE tissue slides, but failed 1 hour hybridization on both slide types.

Results indicate that RF PCR probe designs surpassed the performance of synthesized oligo designed either by direct synthesis or oligo-PCR hybrid. Directly synthesized HER2 oligo probe and oligo-PCR hybrid p53 probe failed 1 hr FFPE, while oligo-PCR hybrid HER2 probe failed all quality evaluation. Based on these data, oligo probe designs were excluded from further evaluation, while RF PCR method was used for the selection of optimal probe designs.

All RF PCR designs passed quality evaluation for 1 hour and overnight hybridization on both lymphocytes and FFPE slides, except some sub-optimal specificity data points observed on lymphocyte slides.

2-step PCR design has several downstream or cleanup options, such as DNA fragment fractionation after sonication, 2nd PCR with aminoallyl-dUTP, and adaptor removal after 2nd PCR. Table 1 lists all the downstream or cleanup steps for each probe designs For both HER2 2-step probes, the different fractionation size, 2nd PCR with aminoallyld-UTP or not, and adaptor removal or not after 2nd PCR did not have a positive impact on the final probe quality.

Same as the three p53 2-step PCR probes, 2nd PCR with aminoallyl-dUTP vs. amination, and adaptor removal or not after 2nd PCR did not impact final probe quality.

The final procedure for Repeat-Free PCR FISH Probe design and preparation is the following.

1: Identify desired coordinates of locus on Genome Browser.
2: Download sequence with repeats masked to N.
3: Enter sequence in Sequence Processing program.
4: Process to identify kilomers of unique sequence, and format for BatchPrimer3
5: Use BatchPrimer3 (web) to identify F and R primers for each kilomer.
6: Order mixed primers in 96-well plate format.
7: PCR in plate using Phusion polymerase, assess by eGel-96.
8: Combine PCR products, fragment by sonication.
9: Blunt sonicated DNA.
10: Ligate Adaptors to make template.
11: PCR with: Adaptor-primers to make bulk DNA for amination, labeling.
OR Adaptor-primers and AAdUTP to make aminated DNA for labeling.
13: Aminate (if needed)
14: Label, formamide treat.

Example 3

This example describes the development of repeat-free probes targeting p16. A map of the p16 probe is shown in FIG. 9. Primer sequences are shown in Table 4.

TABLE 4

SEQ

ID

Name
Sequence
NO.:

17603_1_F
AATGAGACACGTGAGATCTGGAAGG
683

17603_1_R
CAATTTGTTTTGTTTTAGGGCAGGA
684

20736_1_F
ATTTACGGTAGTGGGGGAAGGCATA
685

20736_1_R
AATGCAAGCTACGGGAGAAAGAAAC
686

22171_0_F
TTAATTGTGCTTGAAGAGGGGGTGT
687

22171_0_R
TTATACTTCCCAAAGCATACCACCA
688

22171_545_F
CGTGTTAATTCCCGTGTACTGTTTCA
689

22171_545_R
CGAGTGGCGGAGCTGCTG
690

22171_1090_F
CCTCTAGCCTCTTGAGTCTTCATTGC
691

22171_1090_R
TGAAGTTCAACATTCCCAGAAGCTAA
692

25362_1_F
TCCCAGGTTTATGATTTGAGAGTTCA
693

25362_1_R
GGAACTTAGGAAATAATGAGCCACA
694

26887_0_F
TTCCAACATACACCACAGATTTCCA
695

26887_0_R
GCCATACTTTCCCTATGACACCAAA
696

26887_494_F
GACCGTAACTATTCGGTGCGTTG
697

26887_494_R
TGTGAGAAGTGTGAAGGAGACAGGA
698

31895_1_F
TTATTCCTGAAAACCAGCCTGTGAA
699

31895_1_R
TTGATGGTTGTTACCAATACATGCTC
700

34379_1_F
ACAGATTGTGGTTTAGCCCCGAAGT
701

34379_1_R
TGTTCTCAGCTGATCAAATAGCTACAAA
702

36757_0_F
TGAAGTTATTCATTTAGGTCATCAA
703

36757_0_R
ATTCTGTTATCATTGCCTTTTTGAA
704

36757_584_F
GGTTTTCACTACTGGGAGTGGAGGT
705

36757_584_R
TCCTGGTATGTTCCAAGGTGTTAGG
706

44110_1_F
TCAGTTTTAGTTCGGCCTAGAATGTT
707

44110_1_R
TTGCCTCTGAAAATCTGTAGTAACAAA
708

45454_1_F
CAGTTACCGGTCACAGTGGCTAAAC
709

45454_1_R
TCAAGCACACTTTCTTTCTCCTTGG
710

47238_0_F
CCAAGATCTCGGAACGGCTCT
711

47238_0_R
GTCCTCGACTCACCCCTCCTTT
712

47238_654_F
ACAAGCACCGAGTCCTTTGTGTCTA
713

47238_654_R
ACGGTGTTGGGTAAATTCTGAGTGA
714

53456_1_F
AAGAGAGAAACCCGAAGAACAATGG
715

53456_1_R
CGTTTCTCAATTTCAGCTATCCAAATG
716

54621_1_F
CTCAACTGTTGTTGCCCCTTTAAGT
717

54621_1_R
CATTCATTTG GCTTCATTTTTATCTT
718

55625_1_F
CCAGATCTTCTTGGAATAAATGTCAGG
719

55625_1_R
CACAATGATCTCCCTTGTAAGCTCCT
720

56854_1_F
CCTTTCCCCAATAACATATGCTCTGA
721

56854_1_R
CGGAAATCCCCTTATGACTTGCTAC
722

57838_0_F
AACCGTTACAATTGCTCTCACTCCA
723

57838_0_R
GGTGTTTCTTTAAATGGCTCCACCT
724

57838_736_F
GGGTGGGAAATTGGGTAAGAAAATA
725

57838_736_R
AAAATGATGAACTGTTAAGGAAAAATCA
726

60520_0_F
TGCTCCTTTAAAAATCCCTCATTTG
727

60520_0_R
GAGTGTCGTTAAGTTTACGGCCAAC
728

60520_779_F
GGATTTTTGCTGGGTAAAAGCCTGT
729

60520_779_R
GAAACTTGTGAAGCCCAAGTACTGC
730

60520_1558_F
AAGCCTGCCCAAAGATGCTAGGAC
731

60520_1558_R
TAATTCTACAGGGATTTGGGGGATG
732

60520_2337_F
GGGCTTGTCATTAAACAGGCTGAAC
733

60520_2337_R
TCATATTTGAAAACCAGGTTGAGCAG
734

81987_1_F
AATAGGAGAGCCTGATCATGTGTGG
735

81987_1_R
TCTGCCAGTAGTTTTAAAGGGCTGA
736

87808_0_F
TTTAGAGAAAATAAGTGCTGCTGAGG
737

87808_0_R
GCACTACTGGTTGTTTTAGGCTTTTTC
738

87808_643_F
TGCCTGATAGAAGTCAGACTCTGTGG
739

87808_643_R
GAGCCACCTTTCAATCCCATTCTAC
740

89872_1_F
TAGCAATTCTCATTTGGATTCCTGT
741

89872_1_R
CCTCTCTCCTCTAGGTGGCAAACTC
742

91063_1_F
CTATTTTGCCAGCGCAGATTTGATA
743

91063_1_R
CAACAACAATGCTATCCACACAGATG
744

93683_1_F
TGGTTTTGTACCACTCCCTCTCTCA
745

93683_1_R
AGCCCCTAGACAGCTGGTAGAAGAA
746

96599_1_F
ATATTCAAGCATGAGGAATGGCAAA
747

96599_1_R
TTTCCAAAAGAAAAATACTTCTCCTCA
748

103078_0_F
AAGTCCCAGCTGAAAGGTAACCAAA
749

103078_0_R
AACTGCCTTTGGAAGAAAATTCAGG
750

103078_648_F
GGTTTTCATGAACCTCACATGGACT
751

103078_648_R
CTCGAAATTCCAAGGGCACTGAAAA
752

105020_0_F
GAAAAGCTAGAGTCACATTTTAGTGACC
753

105020_0_R
AAACCAAACAGAATTTCCACAGACC
754

105020_376_F
TCAGTCCAGAGCAAGTGAATACTGC
755

105020_376_R
TTTTTCCTTCGATGGCTGTAACAAT
756

114540_1_F
ATGTAGGCTTGTGCCTGACATTAAA
757

114540_1_R
TTAGGGGAAATTTTAGAGCCAACGA
758

116353_1_F
TGCTGATGAGTGACCAGTTTAGATGA
759

116353_1_R
GCCCACCTTTTAGGTTTGTCGTTAG
760

117676_1_F
CCCACACCTTTGATGTATTTCTCTTTG
761

117676_1_R
GATTTCAGCTCTTCATCACCCACAT
762

121286_1_F
TCTTTTCCAGTGACGTAGTGTGTGG
763

121286_1_R
TCTCTCAGTGAATCATTTTCCTAGGAGTT
764

125649_1_F
TGCTATTATGGCACAATTGATGTTGA
765

125649_1_R
CATGATCTTATTTCAACCAACTCTCAAA
766

132233_1_F
GAGGGAACAACTTGTGGAAAAGTGA
767

132233_1_R
GAAGCATAGGCTAATTCTCATGCAA
768

136934_1_F
TAGGGCTATTTCACAAAAGGGCTGT
769

136934_1_R
GTGTGGCTAGGTCCTTGGGATAAAC
770

141163_1_F
AAGGTCAGTAGGTTCACAGGCCCTA
771

141163_1_R
TTCACTTGAAGATAATGAACATCTTACC
772

149647_1_F
CTGGATTTGCAGTGTTGTGTCCTAT
773

149647_1_R
CAACATTCTGTTCTGAGTGCTGGTG
774

156352_1_F
GAGTAGACAGCCAACCCCCTGTATT
775

156352_1_R
AGCATGTAGGAGAGGTACGGTGGTA
776

159972_1_F
AGCCGTTAGTCAACTCTGAGTGCAG
777

159972_1_R
TGACCAAGCAATGTGCATAGACAAG
778

161650_1_F
CCCAAAACTGAAAGATGAAAACATA
779

161650_1_R
TGCAGAAAGGATCCTATTGTTGGTG
780

168102_1_F
CTCAGAAGCGAGGGAGACTTAAACC
781

168102_1_R
CCCTGGCTCATCTCCATACCTACTC
782

169536_1_F
CCATTCTTCCTGAATAACTTGGCTA
783

169536_1_R
TCAAAGGTTTGAACTTGCCTCTCAC
784

176637_1_F
AACAAAGCAAATTGCAGGCAATAAA
785

176637_1_R
AGCCTGTGGTCATAAAACACCTCAC
786

181078_0_F
GAAGCATAAAGCAATGGACCAGGTT
787

181078_0_R
TTTTCCTCACCTTATGTCAACAAGGA
788

181078_648_F
CTTGGGGTTTCAGGAGAGAAATTGT
789

181078_648_R
TGACACAGACAAACAGGTGACTCAA
790

181078_1296_F
AATTTGTTCTTTCAATTTGGCATGG
791

181078_1296_R
TCAATTTTCTGCATGAATTTCCACT
792

185793_1_F
CATAATGGGCCACATGATTTTTACC
793

185793_1_R
GTAAAGTCAAAAACCTTCCCCATCC
794

187891_0_F
CAACTGCTGAACATAGACCCAAGG
795

187891_0_R
AAATTCATAACATTTTTCCTTTGAA
796

187891_480_F
TTGTTGGAAAGCACATTAGGTGTGT
797

187891_480_R
TCCTGTGAAGAAAGCTATGCAGTTG
798

187891_960_F
AAAATCTTGGTCTTTCCCACAGAGG
799

187891_960_R
AGACTCTTATTGGTGGTTTGTGGAA
800

194665_1_F
GCCAGCATTGAGCTGATAAAGACCT
801

194665_1_R
CAAAAGTGAAAGAAAATGAATGAAACA
802

197665_1_F
CCGGATTTATTTATTGTTCCTGGTTG
803

197665_1_R
TGCCAGGCTCTATATGTGTTTTGTG
804

199017_1_F
TGAAAAGAGTGGTGCTAGATACTTGGA
805

199017_1_R
TCACCTACAACTGATTGGCAGTGTC
806

208772_1_F
GGCCTAAGAATTAGTGAAAATCCCAAC
807

208772_1_R
TCATCCTTCACAGATCAACCTCCTC
808

210208_1_F
CTGAACTCAGAAGGTGAACCACACC
809

210208_1_R
CCTGTAAAAGTCCTCAGGTCAACTCA
810

211384_1_F
TGAGAATTTGTGATTTCCTGGTGTTG
811

211384_1_R
GAAATCCTATAGATAGAAAAGCATTCA
812

212493_1_F
ATATCTCTCTTTGGTTCCCTTTTT
813

212493_1_R
TCCTTAAGAAATCAGTATTCCAATGCAAA
814

214609_1_F
CATCCAAAAGGAGAGTTTGGATTTG
815

214609_1_R
CATGCCTAGAAAATCGTGGCTGATA
816

216869_0_F
AAAAAGGGTGTGGCTTTATTTATTT
817

216869_0_R
CCAACAAATCTGTAGATTGCTGACA
818

216869_500_F
AAAGAAAGCCAAGTTAGGTTCAGACA
819

216869_500_R
TGAGAGGTTGCTGAGGAATTCTTGT
820

222534_0_F
GCCTAGTGGAAAAACATTTCCAAGC
821

222534_0_R
ACAGCTGGACATGCATGCTCTTAAT
822

222534_366_F
TTGCAACTTTCCAGACATTTATTTTC
823

222534_366_R
AGGGTTTCCAAAGTTTTGGTGAATG
824

225452_1_F
TAAAATTTGTTTGGAGGTGGGGTCT
825

225452_1_R
ACCCACAGAAACTTCCATTTCACTG
826

228586_1_F
CACAAGAAAAGGTTTCATGAGATAGG
827

228586_1_R
TG CCATCTAAAATATTGCTCCTACCA
828

PCR of 1200mers with Phusion: To 2 μL each p16RF 5 uM, 18 μL Phusion MM was added and placed on thermocycler with program 98 deg 30 sec, 25× (98° C. 8 sec, 68° C. 30 sec, 72 deg 1 min), 72° C. 10 min, 4° C. Extend PCR: To each well, 2 μL of the same 5 μM primer and 2 μL (80 uL 2×pcrMM+60 μL dNTP+20 μL 1 M MgCl₂) was added and thermocyling was performed: 10× (98° C. 8 sec, 68° C. 30 sec, 72° C. 2 min). Loaded 3 μL to wells of eGel, electrophoresed 8 min. Most wells (68/73) showed strong clean bands at expected (same) MW. However some show closely spaced doublets. Wells of p16RF Phusion PCR were combined to total 1200 μL, split to 3×400 μL in tubes and 40 μL 3M NaAc and 1 mL EtOH was added and tubes were placed at −20° C. Tubes were spun at 15000 rpm at 4 deg for 12 min, pellets were washed in 400 μL 70% EtOH, dried in speedvac, combined, and resuspended in 200 μL water, placed in 65 deg oven 5 min, and spun at 4000 rpm 5 min to sediment polymerase. The supernate was transferred to a new tube, 300 μL PEG rgt was added. After 15 min, the tube was spun at 14000 rpm for 5 min, the pellet was resuspended in 400 μL water. Electrophoresis showed bands consistent with 1200mer mix.

Sonicate p16-1200mer: (reserved 20 μL for use as template) To 380 uL p16-1200mer, 40 uL 3M NaAc was added, the tube was sonicated intensity 3, 30% duty cycle 2×10 min, and 1 mL EtOH was added. The tube was spun at 15000 rpm 4° C. for 10 min, the pellet washed in 400 μL 70% EtOH, dried, and resuspended in 100 μL water. The tubes was then spun at 15000 rpm 4 deg 5 min to sediment residue from sonicator probe. Electrophoresis showed a size range of approximately 50-400 bp.

Fractionation: To 10 μL of the sonicated 1200mer, 40 μL water and 10 μL buffer was added, the tube was mixed and pipetted to spin column 1, spun 14000 rpm 1 min; to effluent added 10 μL buffer, mixed, pipetted to spin column 2, spun 14000 rpm 1 min; to effluent added 10 μL buffer, mixed, pipetted to spin column 3, spun 14000 rpm 1 min. Columns were washed with 2×200 μL wash buffer and eluted with 50 μL elution buffer. Electrophoresis showed size of approximately 100-300 bp.

Blunt p16son and p16s2 using Fast DNA End Repair Kit: To 3 μL of the above in 40 μL water, 5 μL of 10× buffer and 2.5 uL enzyme mix was added and the tube was vortexed and placed in 20° C. bath for 20 min. An Inverse PCR purification kit (Life Technologies) was used according to directions to isolate products in 50 μL buffer.

Ligate Adaptors to p16sonB, p16s2B: One μL each of 1000 μM PadBsPD1, CadBsPD1, PadEcoR1, CadEcoR1, 5 μL 10× Ligase Buffer, 5 μL 50% PEG4000, 5 μL 7/28(p16sonB, p16s2B) was added to each tube, the tube was vortexed and 1 μL Ligase was added. An Inverse PCR purification kit was used to isolate products.

PCR of adapted p16: To wells of strip added (8, 8, 2, 8, 2) μL water and (1 μL each 20 μM, 2, 8 μL 20 uM PadBsPD1; 2, 8 μL 20 uM PadEcoR1). To wells added 10 μL of 50 μL DreamTaq Green 2×pcrMM+2 μL of the above probe. Thermocycling was performed with program 25× (95° C. 30 sec, 55° C. 30 sec, 72° C. 30 sec). Analysis showed product bands from p16sonA template slightly broader than those from p16p2A. For both, single primer at 2 μM gave yield similar to 8 μM; no difference between PadBsPD1 and PadEcoR1 primers. PadEcoR1 gave best overall performance.

Prep PCR of adapted p16 with PadEcoR1 single primer 5 μM. To wells of strip added 100 μL (400 μL water+4 μL 1000 μM PadEcoR1+2 μL (p16sonA, p16s2A)+200 μL DreamTaq 2×pcrMM), capped, placed on thermocycler with program 25× (95° C. 30 sec, 55° C. 30 sec, 72° C. 30 sec). Next, an additional 3.0 (30 μL 25 mM dNTP+10 μL 1M MgCl₂+8 μL 1000 μM PadEcoR1) was added to each well and thermocycling was performed with program 10× (94° C. 30 sec, 55° C. 30 sec, 72° C. 2 min). Product was precipitated and resuspended as above.

Aminate p16 preps: Transferred 50 μL of above p16son to tube and used speedvac to reduce volume to 20 μL. Placed this and remaining 20 μL 7/29 p16sp, p16sp2p in boiling water bath 1 min, then to each added 180 μL (500 μL water+300 μL TFA+174 μL ethylenediamine+95 mg Na₂S₂O₃and placed in 65° C. water bath for 15 minutes. Next, solutions were desalted into water and 450 μL of each nanodrop was collected. TMED/NaCl was added, the tubes were placed in 65° C. bath 5 min, 50 μL 3M NaAc and 1 mL isopropanol were added. The resulting solutions were precipitated and resuspended in water.

Label aminated p16 products with TAMRA, CR6G: One μL 1M NaOH was added to above; after 1 min 25 μL (75 μL TMED/NaCl+75 μL DMSO), were added, the tubes were vortexed, and 2 μL (100 mM TAMRA, 100 mM CR6G) was added. Resulting solutions were precipitated and resuspended in water+15 μL 20×SSC and 105 μL formamide to give 150 μL at 2×SSC. Resulting solutions were purified as above.

All publications and patents mentioned in the present application and/or listed below are herein incorporated by reference. Various modification, recombination, and variation of the described features and embodiments will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although specific embodiments have been described, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes and embodiments that are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

HYBRIDIZATION PROBES AND METHODS

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Abstract

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Provisional Applications (1)