Patent Grant
8722336
The invention in part pertains to methods for detecting specific alleles in a mixed nucleic acid sample. Methods provided herein can be used to detect the presence or absence of fetal nucleic acid in a maternal sample.
The analysis of circulating nucleic acids has revealed applications in the non-invasive diagnosis, monitoring, and prognostication of many clinical conditions. For example, for prenatal applications, circulating fetal-specific sequences have been detected and constitute a fraction of the total DNA in maternal plasma. The diagnostic reliability of circulating DNA analysis depends on the fractional concentration of the targeted sequence, the analytical sensitivity, and the specificity. The robust discrimination of sequence differences (e.g., single-nucleotide polymorphisms, or SNPs) between circulating DNA species is technically challenging and demands the adoption of highly sensitive and specific analytical methods.
Current techniques to detect sequence differences in a DNA sample include allele-specific PCR, restriction digest and Southern blot hybridization, restriction endonuclease-mediated selective-PCR (REMS-PCR), and competitive PCR methods involving the use of fluorescent detection probes. Currently available techniques present several disadvantages. For allele-specific PCR, it is often difficult to design assays with a high degree of allele specificity (Nasis et al. Clin Chem. 2004 April; 50(4):694-701). Restriction digest/Southern blot methods require higher amounts of DNA template than the method provided herein, and lack the sensitivity to detect polymorphic sequences comprising a low relative proportion of total DNA. Restriction endonuclease-mediated selective-PCR (REMS-PCR) has the drawback of requiring a thermostable restriction enzyme that cleaves the wild-type allele. REMS-PCR is described in U.S. Pat. No. 6,261,768, which is hereby incorporated by reference. Use of the technique may not always be possible, and this requirement limits the general utility of the REMS-PCR approach. Competitive PCR lacks the sensitivity to detect polymorphic sequences comprising a low relative proportion (<5%) of total DNA. Competitive PCR with allele-specific fluorescent probes lacks the ability to multiplex assays higher than 2-3 assays in a single tube format. In addition, similar methods utilizing methylation differences between DNA species (for example, US Patent Application Publication No. 20070059707, entitled, “Methods for prenatal diagnosis of chromosomal abnormalities”, which is hereby incorporated by reference) are not effective at low copy numbers of genomic DNA.
The invention in part provides sequence-specific cleavage of nucleic acid to selectively enrich for a particular target nucleic acid. Polymorphic loci are chosen such that only one allele at the polymorphic locus is cleaved by a given cleavage agent, such as a restriction endonuclease. Oligonucleotide primer pairs designed to flank the polymorphism allow amplification of the polymorphic region, or amplicon, by amplification (e.g., PCR). Prior to or during amplification, nucleic acid samples are incubated with the given restriction endonuclease. In some embodiments, the cleavage agent is introduced prior to amplification. This approach results in cleavage of the polymorphic allele or sequence comprising the polymorphic allele that is recognized by the restriction endonuclease, if this allele is present. Cleavage of any template nucleic acid within the amplicon sequence (i.e., between primer pairs) prevents PCR amplification of this template. Therefore, if only one allele of a polymorphism is recognized by the cleavage agent and the corresponding nucleic acid sequence is cleaved by the restriction endonuclease, the relative percentage of the amplifiable alternate polymorphic allele is increased in a manner dependent on the efficiency and specificity of the restriction endonuclease activity. After amplification, the amplified polymorphic alleles can be genotyped or otherwise detected or discriminated by any method known in the art (e.g., using Sequenom's MassARRAY® technology or by RT-PCR).
In some embodiments, the invention in part provides a method for detecting the presence or absence of a target allele at a polymorphic locus in a sample, where the sample contains nucleic acid, which comprises: cleaving a nucleic acid comprising a non-target allele at or near the polymorphic locus with a cleavage agent that recognizes and cleaves the non-target allele, but not the target allele; amplifying uncleaved nucleic acid but not cleaved nucleic acid; and analyzing the amplification products from the previous step to determine the presence or absence of the target allele. In certain embodiments, the method also comprises first obtaining a sample suspected of comprising nucleic acid with target and non-target alleles. In some embodiments, the method is used to distinguish between two individuals, for example, between a mother and a fetus, where the sample comprises both maternal and fetal nucleic acid. Optionally, the method may be used to quantify the target nucleic acid relative to the non-target nucleic acid.
The invention also in part provides methods for enriching for target nucleic acid, comprising cleaving nucleic acid comprising a non-target allele with a restriction endonuclease that recognizes the nucleic acid comprising the non-target allele but not the target allele; and amplifying uncleaved nucleic acid but not cleaved nucleic acid, where the uncleaved, amplified nucleic acid represents enriched target nucleic acid relative to non-target nucleic acid. In some embodiments, methods provided herein may be utilized to determine the presence or absence of target nucleic acid in a background of non-target nucleic acid. In certain embodiments, the amplification products can be analyzed to diagnose, monitor or prognose a clinical condition. Likewise, the amplification products can be analyzed to assist in the diagnosis, prognosis or monitoring of a clinical condition or chromosomal abnormality. Nucleic acid may be selected such that it comprises an allele having a polymorphic site that is susceptible to selective digestion by a cleavage agent, for example.
Methods provided herein are useful for analyzing nucleic acid including, but not limited to, DNA, RNA, mRNA, oligonucleosomal, mitochondrial, epigenetically-modified, single-stranded, double-stranded, circular, plasmid, cosmid, yeast artificial chromosomes, artificial or man-made DNA, including unique DNA sequences, and DNA that has been reverse transcribed from an RNA sample, such as cDNA, and combinations thereof. In some embodiments, methods provided herein are used to detect or selectively enrich RNA.
A nucleic acid may also be characterized as target nucleic acid or non-target nucleic acid, where target nucleic comprises the target allele and non-target nucleic acid comprises the non-target allele. In some embodiments, the target nucleic acid comprises the paternal allele and the non-target nucleic acid comprises the maternal allele. In certain embodiments, the nucleic acid is cell-free nucleic acid or partially cell-free nucleic acid. In some embodiments, the target nucleic acid is apoptotic or partially apoptotic. In certain embodiments, the target nucleic acid is less than 2000, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, 50, 40 or less base pairs in length.
Methods provided herein may be used to detect target nucleic acid in a biological sample. In some embodiments, the biological sample is from an animal, often a human. In certain embodiments, the biological sample is selected from the group of whole blood, serum, plasma, umbilical cord blood, chorionic villi, amniotic fluid, cerbrospinal fluid, spinal fluid, lavage fluid, biopsy sample, urine, feces, sputum, saliva, nasal mucous, prostate fluid, semen, lymphatic fluid, bile, tears, sweat, breast milk, breast fluid, embryonic cells and fetal cells, and mixture thereof. In some embodiments, the sample is from a crime scene (e.g., used for forensic analysis). In certain embodiments, the biological sample is obtained through non-invasive means, for example, a blood draw from a pregnant female. In another some embodiments, the biological sample is cell-free. In certain embodiments, the sample is a previously isolated sample of nucleic acids.
In some embodiments, the invention in part provides a method for detecting the presence or absence of fetal nucleic acid in a maternal sample, where the sample contains nucleic acid, which comprises: cleaving nucleic acid comprising a maternal allele with a restriction endonuclease that recognizes and cleaves the nucleic acid comprising the maternal allele but not the paternal allele; amplifying uncleaved nucleic acid but not cleaved nucleic acid; and analyzing the amplification products from the previous step to determine the presence or absence of fetal nucleic acid. In certain embodiments, the sample comprises a mixture of nucleic acids. For example, the mixture may comprise nucleic acid from different species or from different individuals. In some embodiments, the sample is from a pregnant female. Samples can be collected from human females at 1-4, 4-8, 8-12, 12-16, 16-20, 20-24, 24-28, 28-32, 32-36, 36-40, or 40-44 weeks of fetal gestation, and sometimes between 5-28 weeks of fetal gestation. In certain embodiments, methods provided herein may be used to detect the presence or absence of fetal Y-chromosome nucleic acid, thereby determining the sex of the fetus.
In some embodiments, the target nucleic acid comprises a paternal allele. In certain embodiments, the mother is homozygous at the polymorphic site and the fetus is heterozygous at the polymorphic site. In the case when the mother is homozygous at the polymorphic site and the fetus is heterozygous at the polymorphic site, the polymorphic site is considered informative (e.g., see
In certain embodiments, the sample contains nucleic acid from two different individuals. Such instances include, but are not limited to, organ transplant recipients, transfusion recipients, and forensic applications.
In certain embodiments, the sample is from an individual suspected of suffering from a disease, and the non-target allele is a wild-type allele that is selectively cleaved in order to enrich for a disease-related point mutation. In certain embodiments, the disease is cancer. The ras proto-oncogenes, K-ras, N-ras, and H-ras, and the p53 tumor suppressor gene are examples of genes which are frequently mutated in human cancers. Specific mutations in these genes leads to activation or increased transforming potential.
The invention also in part provides methods useful for detecting rare alleles or low copy number alleles. In some embodiments, the target allele is undetectable by conventional or unmodified genotyping methods if the non-target allele is not selectively cleaved. In certain embodiments, the target allele is not detectable unless it is selectively enriched, for example, by methods provided herein. In certain embodiments, the target allele concentration (e.g., allele concentration in a sample) is about 0.1% to about 40%, e.g., about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35%, of total nucleic acid (e.g., total nucleic acid in a composition or sample), or is less than one of the foregoing percentages. Total nucleic acid includes maternal nucleic acid and any fetal nucleic acid, and total nucleic acid includes non-target allele and any target allele. When fetal nucleic acid is present, target allele is about 50% of the fetal nucleic acid, and non-target allele often includes the other about 50% of the fetal nucleic acid and all maternal nucleic acid, in some embodiments. In certain embodiments, the target nucleic acid number is about 1 to about 5,000 molecules, e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900 or 1000 molecules, or is less than one of the foregoing numbers of molecules. In certain embodiments, the target allele is a mutation, and the non-target allele is the wild-type allele. In certain embodiments, the target allele may be either a somatic or germline mutation. In certain embodiments, another allele or sequence identifier in the same amplicon as the polymorphic locus may be detected. For example, a sequence comprising a target allele may be selectively enriched using methods provided herein, and another sequence identifier may be detected by any method known in the art.
In certain embodiments, there are no other polymorphic loci within the amplicon that may be recognized by the cleavage agent. For example, there is only one polymorphic locus in the amplicon recognized by the cleavage agent in some embodiments.
In certain embodiments, the method optionally comprises first isolating nucleic acid from the sample. DNA isolation from blood, plasma, or serum of the pregnant mother can be performed using any method known to one skilled in the art. Any standard DNA isolation technique can be used to isolate the fetal DNA and the maternal DNA including, but not limited to, QIAamp DNA Blood Midi Kit supplied by QIAGEN. Other standard methods of DNA isolation are described, for example, in (Sambrook et al., Molecular Biology: A laboratory Approach, Cold Spring Harbor, N.Y. 1989; Ausubel, et al., Current protocols in Molecular Biology, Greene Publishing, Y, 1995). A method for isolation of plasma DNA is described in Chiu et al., 2001, Clin. Chem. 47: 1607-1613, which is herein incorporated by reference in its entirety. Other suitable methods are provided in Example 2 of PCT International Application Publication Number 2007/028155, filed on Sep. 1, 2006.
Methods described herein allow for the use of any cleavage agent capable of distinguishing between two different sequences, and cleaving somewhere within the amplicon sequence thereby preventing amplification of the cleaved sequence. The difference between the sequences may be the result of different alleles at one or more polymorphic sites within the sequence. In another example, the difference between the sequences may be the result of two homologous sequences, for example, between paralogous genes or between highly homologous genes such as the RhD gene, which encodes the D polypeptide, and the RHCE gene, which encodes the CcEe polypeptide. An example of a cleavage agent is a restriction enzyme, also referred to as a restriction endonuclease. Multiple restriction endonucleases (available from various vendors) may be selected that correspond to appropriate sequence differences. In some embodiments, the restriction enzyme is a thermostable restriction enzyme. In certain embodiments, the restriction enzyme is Tsp509I. In certain embodiments, a step is added to end the cleaving activity of the cleavage agent, for example, by introducing a protease and/or high temperature prior to amplification.
A restriction endonuclease may be added prior to or during amplification, for example, during an incubation step. In some embodiments, the restriction endonuclease is added less than 5 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes or 120 or more minutes before amplification. Incubation time may be shortened if additional units of restriction enzyme are added to the reaction. Conversely, longer incubation times are often used to allow a reaction to proceed to completion with fewer units of enzyme. This is contingent on how long a particular enzyme can survive (maintain activity) in a reaction. Some enzymes survive for long periods (>16 hours) while others survive only an hour or less in a reaction. In certain embodiments, the restriction enzyme digests greater than 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the non-target nucleic acid. However, if digestion of non-target nucleic acid of less than 40% allows for useful enrichment of target nucleic acid, it is within the scope of the invention. In certain embodiments, the restriction enzyme digests substantially all of the non-target nucleic acid. In certain embodiments, the restriction endonuclease is a thermostable restriction endonuclease. Examples of thermostable endonucleases include, but are not limited to, Bst NI, Bsl I, Tru 9I and Tsp 509 I. In certain embodiments, the cleavage agent is not thermostable, especially when the digestion occurs prior to the amplification step. In some embodiments, the cleavage agent is thermostable and a majority of the digestion of the non-target nucleic acid occurs prior to the amplification step during a pre-incubation step. In certain embodiments, the restriction enzyme digests greater than 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the non-target nucleic acid prior to amplification. In another embodiment, one or more incubation steps may be introduced during thermal cycling. Incubation steps are ideally at the optimal temperature for digestion to occur. For example, for Tsp509I the incubation temperature may be 65 degrees C. In certain embodiments, a step is introduced to prevent or to reduce digestion during the amplification step, for example, by introducing a protease to disable a cleavage agent that is a protein.
In some embodiments, the units of restriction enzyme added to the sample is 0.10, 0.25, 0.50, 0.75, 1.0, 2.0 or more. Note that DNA substrates are digested at varying rates, therefore, the actual number of units required for a complete or substantially complete digestion may vary from assay to assay.
In certain embodiments, only one restriction endonuclease is used to digest one or more non-target alleles in a single reaction. For example, a multiplexed assay may be designed where a single restriction endonuclease performs multiple (e.g., greater than 5, 10, 15, 20, 25, 50, 100) digestions across the genome. In certain embodiments, more than one restriction endonuclease (e.g., greater than or equal to 2, 3, 4, 5, 6, 7, 8, 9, 10) is used to make multiple (e.g., greater than 5, 10, 15, 20, 25, 50, 100) digestions across the genome.
Amplification may be performed after or during the cleavage of the non-target allele, and prior to the detection of the target allele. In some embodiments, amplification is performed after cleavage of the non-target allele. Amplification can be performed by any method known in the art, including but not limited to polymerase chain reaction (PCR), ligase chain reaction, transcription-based amplification, restriction amplification, or rolling circle amplification, using primers that anneal to the selected fetal DNA regions. Oligonucleotide primers are selected such that they anneal to the sequence to be amplified. In some embodiments, primers are designed such that one or both primers of the primer pair contain sequence recognizable by one or more restriction endonucleases.
Following amplification, the relative enrichment of the target allele in the sample allows accurate detection of allele frequencies using practically any method of nucleic acid detection known in the art. For example, any of the following methods may be used, including, but not limited to, primer extension or microsequencing methods, ligase sequence determination methods, mismatch sequence determination methods, microarray sequence determination methods, restriction fragment length polymorphism (RFLP) procedures, PCR-based assays (e.g., TAQMAN® PCR System (Applied Biosystems)), nucleotide sequencing methods, hybridization methods, conventional dot blot analyses, single strand conformational polymorphism analysis (SSCP), denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis, mismatch cleavage detection, detection by mass spectrometry, real time-PCR and pyrosequencing.
Methods provided herein may also be multiplexed at high levels in a single reaction. For example, one or more alleles can be detected simultaneously. Multiplexing embodiments are particularly important when the genotype at a polymorphic locus is not known. In some instances, for example when the mother is heterozygous at the polymorphic locus, the assay may not be informative. See
In certain embodiments, the invention in part provides a method for quantifying a target allele at a polymorphic locus in a sample, where the sample contains nucleic acid, that comprises: digesting nucleic acid containing a maternal allele at the polymorphic locus with an enzyme, such as a restriction endonuclease, that selectively digests the maternal allele, where the selective digestion yields a DNA sample enriched for fetal DNA; determining the maternal or paternal allele frequency using polymorphic markers within the amplicon, and comparing the paternal or maternal allele frequency to a control DNA sample. In some embodiments, a difference in allele frequency is indicative of a chromosomal abnormality. In certain embodiments, the control DNA sample is a competitor oligonucleotide that is introduced to the assay in known quantities.
In certain embodiments, the present invention provides a kit for detecting the presence or absence of target nucleic acid. One component of the kit is primers for amplifying the region of interest. Another component of the kit comprises probes for discriminating between the different alleles of each nucleic acid species.
Certain non-limiting embodiments of the invention are further described in the following Brief Description of the Drawings, Detailed Description and claims.
It has been determined in the fields of biology and diagnostics that certain nucleic acids are present at very low concentrations in humans. In particular, fetal DNA has been found to exist in maternal plasma (Lo et al. Lancet. 1997 Aug. 16; 350(9076):485-7). This discovery has facilitated the development of non-invasive prenatal diagnostic approaches based simply on the analysis of a maternal blood sample (Lo et al. Am J Hum Genet. 1998 April; 62(4):768-75). The non-invasive nature of maternal plasma-based approaches represents a major advantage over conventional methods of prenatal diagnosis, such as amniocentesis and chorionic villus sampling, which are associated with a small but finite risk of fetal loss. However, a technical challenge experienced by many workers in the field relates to the ability to discriminate the relatively small amount of fetal DNA from the coexisting background of maternal DNA in maternal plasma. During pregnancy, fetal DNA amounts to approximately 3-6% of the total DNA in maternal plasma. Hence, the diagnostic reliability of fetal DNA analysis in maternal plasma generally has depended on the accurate detection of fetal-specific markers.
Methods described herein solve this problem by enriching, relatively, the amount of low copy number nucleic acid before detecting or quantifying the alleles present in the sample. In the case of prenatal diagnostics, the use of restriction endonuclease enhanced polymorphic sequence detection allows for the selective, sensitive detection of fetal nucleic acid from maternal samples. The fetal DNA in the maternal plasma sample is selectively enriched before detecting the alleles present in the maternal sample. To enrich for fetal DNA present in plasma of the mother to allow accurate detection of fetal alleles present in the sample, methods provided herein allow for the cleavage of maternal nucleic acid or nucleic acid of maternal origin. Thus, the maternal DNA can be substantially reduced, masked, or destroyed completely, and the sample is left with DNA enriched for DNA of fetal origin. The selective reduction of maternal DNA can be performed using one or more enzymes, such as restriction endonucleases, which selectively digest nucleic acids which comprise maternal alleles.
The term “sample” as used herein refers to a composition, specimen or culture (e.g., microbiological cultures) that includes nucleic acids. The term “sample” includes biological and environmental samples. A sample may include a specimen of synthetic origin. Biological samples include whole blood, serum, plasma, umbilical cord blood, chorionic villi, amniotic fluid, cerbrospinal fluid, spinal fluid, lavage fluid (e.g., bronchoalveolar, gastric, peritoneal, ductal, ear, athroscopic), biopsy sample, urine, feces, sputum, saliva, nasal mucous, prostate fluid, semen, lymphatic fluid, bile, tears, sweat, breast milk, breast fluid, embryonic cells and fetal cells. A biological sample can be maternal blood, including maternal plasma or serum. In some circumstances, a biological sample is acellular. In other circumstances, a biological sample does contain cellular elements or cellular remnants in maternal blood. In some embodiments, a nucleic acid sample is, or is obtained from, an extracellular or acellular composition (e.g., blood plasma, blood serum, urine).
In some embodiments, a sample comprises a mixture of nucleic acids. For example, the mixture may comprise nucleic acid from different species or from different individuals. In some embodiments, a sample is from a pregnant female or a female suspected of being pregnant. In certain embodiments, the sample is procured through non-invasive means (e.g., a blood draw). In some embodiments the sample is from any animal, including but not limited to, human, non-human, mammal, reptile, cattle, cat, dog, goat, swine, pig, monkey, ape, gorilla, bull, cow, bear, horse, sheep, poultry, mouse, rat, fish, dolphin, whale, and shark, or any animal or organism that may be tested for the presence of target nucleic acid.
In some embodiments, the biological sample is blood, and sometimes plasma. As used herein, the term “blood” encompasses whole blood or any fractions of blood, such as serum and plasma as conventionally defined. Blood plasma refers to the fraction of whole blood resulting from centrifugation of blood treated with anticoagulants. Blood serum refers to the watery portion of fluid remaining after a blood sample has coagulated. Environmental samples include environmental material such as surface matter, soil, water, crime scene samples, and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable and non-disposable items. These examples are not to be construed as limiting the sample types applicable to the present invention.
The term “non-invasive” as used herein refers to a method for collecting a sample that poses minimal risk to an individual (e.g., the mother, fetus, victim, etc.). An example of a non-invasive method is a blood draw; whereas examples of invasive methods include amniocentesis and chorionic villus sampling, both of which constitute a finite risk to the fetus.
The terms “target” or “target nucleic acid” as used herein refer to any molecule whose presence is to be detected or measured or whose function, interactions or properties are to be studied, where target nucleic comprises the target allele and non-target nucleic acid comprises the non-target allele. Fetal nucleic acid may comprise both target nucleic acid and non-target nucleic when the fetus is heterozygous at a polymorphic locus. Other examples of target nucleic acid include, but are not limited to, trace nucleic acid, mutated nucleic acid, viral nucleic acid and transplant nucleic acid.
The terms “nucleic acid” and “nucleic acid molecule” may be used interchangeably herein. The terms refer to oligonucleotides, oligos, polynucleotides, deoxyribonucleotide (DNA), genomic DNA, mitochondrial DNA (mtDNA), complementary DNA (cDNA), bacterial DNA, viral DNA, viral RNA, RNA, message RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), siRNA, catalytic RNA, clones, plasmids, M13, P1, cosmid, bacteria artificial chromosome (BAC), yeast artificial chromosome (YAC), amplified nucleic acid, amplicon, PCR product and other types of amplified nucleic acid, RNA/DNA hybrids and polyamide nucleic acids (PNAs), all of which can be in either single- or double-stranded form, and unless otherwise limited, would encompass known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides and combinations and/or mixtures thereof. Thus, the term “nucleotides” refers to both naturally-occurring and modified/non-naturally-occurring nucleotides, including nucleoside tri, di, and monophosphates as well as monophosphate monomers present within polynucleic acid or oligonucleotide. A nucleotide may also be a ribo; 2′-deoxy; 2′,3′-deoxy as well as a vast array of other nucleotide mimics that are well-known in the art. Mimics include chain-terminating nucleotides, such as 3′-O-methyl, halogenated base or sugar substitutions; alternative sugar structures including nonsugar, alkyl ring structures; alternative bases including inosine; deaza-modified; chi, and psi, linker-modified; mass label-modified; phosphodiester modifications or replacements including phosphorothioate, methylphosphonate, boranophosphate, amide, ester, ether; and a basic or complete internucleotide replacements, including cleavage linkages such a photocleavable nitrophenyl moieties.
In the case of RNA, an RNA may be placentally-expressed RNA in maternal plasma. Background maternal RNA may be selectively digested according to methods provided herein. Also, methods herein may further comprise an additional step of discriminating alleles of RNA which involves reverse transcriptase polymerase chain reaction (RT-PCR). In certain embodiments, fetal RNA may be extracted from maternal body fluids, sometimes whole blood, and sometimes plasma or serum using e.g. RNA extraction methods such as, but not limited to, gelatin extraction method; silica, glass bead, or diatom extraction method; guanidinium thiocyanate acid-phenol based extraction methods; guanidinium thiocyanate acid based extraction methods; guanidine-hydrochloride based extraction methods; methods using centrifugation through cesium chloride or similar gradients; phenol-chloroform based extraction methods; and/or other available RNA extraction methods, as are known in the art for use in extraction of intracellular RNA, including commercially available RNA extraction methods, e.g. by using or adapting or modifying methods of Boom et al. (1990, J. Clin. Microbiol. 28: 495-503); Cheung et al. (1994, J. Clin. Microbiol. 32: 2593-2597); Boom et al. (1991, J. Clin. Microbiol. 29: 1804-1811); Chomczynski and Sacchi (1987, Analytical Biochem. 162: 156-159); Chomczynski, (1993, Biotech. 15: 532-537); Chomczynski and Mackey (1995, Biotechniques 19: 942-945); Chomczynski and Mackey (1995, Anal. Biochem. 225: 163-164); Chirgwin et al. (1979, Biochem. 18: 5294-5299); Fournie et al. (1986 Anal. Biochem. 158: 250-256); and W097/35589.
The term “amplification reaction” as used herein refers to any in vitro means for multiplying the copies of nucleic acid. “Amplifying” as used herein refers to a step of submitting a sample to conditions sufficient to allow for amplification. Components of an amplification reaction may include, but are not limited to, e.g., primers, a polynucleotide template, polymerase, nucleotides, dNTPs and the like. The term “amplifying” typically refers to an “exponential” increase in target nucleic acid. However, “amplifying” as used herein can also refer to linear increases in the numbers of a select target sequence of nucleic acid, but is different than a one-time, single primer extension step. “Polymerase chain reaction” or “PCR” as used herein refers to a method whereby a specific segment or subsequence of a target double-stranded DNA, is amplified in a geometric progression. PCR is well known to those of skill in the art; see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202; and PCR Protocols: A Guide to Methods and Applications, Innis et al., eds, 1990.
“Oligonucleotide” as used herein refers to linear oligomers of natural or modified nucleosidic monomers linked by phosphodiester bonds or analogs thereof. Oligonucleotides include deoxyribonucleosides, ribonucleosides, anomeric forms thereof, peptide nucleic acids (PNAs), and the like, capable of specifically binding to a target nucleic acid. Usually monomers are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a few monomeric units, e.g., 3-4, to several tens of monomeric units, e.g., 40-60. Whenever an oligonucleotide is represented by a sequence of letters, such as “ATGCCTG,” it will be understood that the nucleotides are in 5′-3′ order from left to right and that “A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine, “T” denotes deoxythymidine, and “U” denotes the ribonucleoside, uridine, unless otherwise noted. Oligonucleotides often comprise the four natural deoxynucleotides; however, they may also comprise ribonucleosides or non-natural nucleotide analogs. Where an enzyme has specific oligonucleotide or polynucleotide substrate requirements for activity, e.g., single stranded DNA, RNA/DNA duplex, or the like, then selection of appropriate composition for the oligonucleotide or polynucleotide substrates is well within the knowledge of one of ordinary skill.
As used herein “oligonucleotide primer”, or simply “primer”, refers to a polynucleotide sequence that hybridizes to a sequence on a nucleic acid template and facilitates the amplification of the nucleic acid template, or otherwise plays a role in the detection of the nucleic acid molecule. In amplification embodiments, an oligonucleotide primer serves as a point of initiation of nucleic acid synthesis. Primers can be of a variety of lengths and are often less than 50 nucleotides in length, for example 12-25 nucleotides, in length. The length and sequences of primers for use in PCR can be designed based on principles known to those of skill in the art.
The term “template” refers to any nucleic acid molecule that can be used for amplification in methods described herein. RNA or DNA that is not naturally double stranded can be made into double stranded DNA so as to be used as template DNA. Any double stranded DNA or preparation containing multiple, different double stranded DNA molecules can be used as template DNA to amplify a locus or loci of interest contained in the template DNA.
The term “amplicon” as used herein refers to amplified DNA that has been “copied” once or multiple times, e.g. by polymerase chain reaction. The amplicon sequence falls between the amplification primers.
The term “polymorphic locus” as used herein refers to a nucleic acid region that comprises a polymorphism. The nucleic acid region may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or more nucleotides in length.
The term “polymorphism” as used herein refers to an allelic variant. Polymorphisms can include single nucleotide polymorphisms (SNP's) as well as simple sequence length polymorphisms, for example. A polymorphism can be due to one or more nucleotide substitutions at one allele in comparison to another allele or can be due to an insertion or deletion, duplication, inversion and other alterations of one or more nucleotides. Certain polymorphisms include, but are not limited to, restriction fragment length polymorphisms (RFLPs), insertions/deletions, short tandem repeats, such as di-, tri- or tetra-nucleotide repeats (STRs), and the like. As used herein, the term “polymorphism” includes epigenetic variants, as long as cleavage by non-epigenetic-specific cleavage agents is utilized.
The term “allele” as used herein is one of several alternate forms of a gene or non-coding regions of DNA that occupy the same position on a chromosome. The term allele can be used to describe DNA from any organism including but not limited to bacteria, viruses, fungi, protozoa, molds, yeasts, plants, humans, non-humans, animals, and archeabacteria.
Alleles can have an identical sequence or can vary by a single nucleotide or more than one nucleotide. With regard to organisms that have two copies of each chromosome, if both chromosomes have the same allele, the condition is referred to as homozygous. If the alleles at the two chromosomes are different, the condition is referred to as heterozygous. For example, if the locus of interest is SNP X on chromosome 1, and the maternal chromosome contains an adenine at SNP X (A allele) and the paternal chromosome contains a guanine at SNP X (G allele), the individual is heterozygous A/G at SNP X.
As used herein, the term “mutant alleles” may refer to variant alleles that are associated with a disease state, e.g., cancer. The term “sequence identifier” as used herein refers to any sequence difference that exists between two sequences that can be used to differentiate the sequences. In some embodiments, the sequence identifier does not include methylation differences.
As used herein, the term “genotype” refers to the identity of the alleles or non-homologous variants present in an individual or sample. The term “genotyping a sample” or “genotyping an individual” refers to determining a specific allele or specific nucleotide(s) or polymorphism(s) in a sample or carried by an individual at particular region(s).
The term “selectively” as used herein does not suggest an absolute event, but instead a preferential event. For example, “selectively cleaved” is used to indicate one sequence (for example, the non-target sequence) is preferentially cleaved or digested over another sequence (for example, the target sequence). However, some of a target sequence may also be cleaved due to a lack of specificity with the cleavage agent or other variables introduced during the cleavage process.
The term “cleavage agent” as used herein refers to any means that is capable of differentially cleaving two or more sequences based on a sequence difference that exists between the two or more sequences. The cleavage agent may be an enzyme in some embodiments. The cleavage agent may be natural, synthetic, unmodified or modified. In some embodiments, the cleavage agent is a restriction endonuclease. Restriction endonucleases, alternatively called restriction enzymes, are a class of bacterial enzymes that cut or digest DNA at specific sites. Type I restriction endonucleases occur as a complex with the methylase and a polypeptide that binds to the recognition site on DNA. They are often not very specific and cut at a remote site. Type II restriction endonucleases are the classic experimental tools. They have very specific recognition and cutting sites. The recognition sites are short, 4-8 nucleotides, and are usually palindromic sequences. Because both strands have the same sequence running in opposite directions the enzymes make double-stranded breaks, which, if the site of cleavage is off-center, generates fragments with short single-stranded tails; these can hybridize to the tails of other fragments and are called sticky ends. They are generally named according to the bacterium from which they were isolated (first letter of genus name and the first two letters of the specific name). The bacterial strain is identified next and multiple enzymes are given Roman numerals. For example the two enzymes isolated from the R strain of E. coli are designated Eco RI and Eco RII. In some embodiments, the restriction enzyme is a type II restriction endonuclease. In another some embodiments, the restriction enzyme is thermostable.
The term “chromosomal abnormality” as used herein refers to a deviation between the structure of the subject chromosome and a normal homologous chromosome. The term “normal” refers to the predominate karyotype or banding pattern found in healthy individuals of a particular species. A chromosomal abnormality can be numerical or structural, and includes but is not limited to aneuploidy, polyploidy, inversion, a trisomy, a monosomy, duplication, deletion, deletion of a part of a chromosome, addition, addition of a part of chromosome, insertion, a fragment of a chromosome, a region of a chromosome, chromosomal rearrangement, and translocation. A chromosomal abnormality can be correlated with presence of a pathological condition or with a predisposition to develop a pathological condition.
Uses and Advantages Associated with Methods Described Herein
The invention in part provides nucleic acid-based assays that are particularly useful for non-invasive prenatal testing. Methods provided herein may be used, inter alia, to determine the presence of fetal nucleic acid in a sample, to determine the amount of fetal nucleic acid in a sample, to determine the sex of a fetus, and to enrich for a target nucleic acid sequence. The invention in part may be combined with other prenatal methods, such as those described in U.S. application Ser. No. 12/027,954, filed Feb. 7, 2008; PCT Application No. PCT/US07/69991, filed May 30, 2007; PCT Application No. PCT/US07/071232, filed Jun. 15, 2007; PCT Patent Publication Numbers WO 2009/032779 and WO 2009/032781, both filed Aug. 28, 2008, PCT Patent Publication Number WO 2008/118988, filed Mar. 26, 2008, and PCT Patent Application Number PCT/EP05/012707, filed Nov. 28, 2005; or any of the prenatal diagnostic (both invasive and non-invasive) methods disclosed in PCT Patent Publication No. WO 2008/157264, filed on Jun. 12, 2008, all of which are hereby incorporated by reference.
The invention in part may be used to more accurately detect fetal DNA using high frequency polymorphisms that match the criteria provided herein. These polymorphisms are alternatively called fetal identifiers. The criteria includes one or more of the following:
1) One allele of the SNP is recognized by the cleavage agent;
2) The alternate SNP allele is not recognized by the same cleavage agent;
3) No other sites for the cleavage are found +/−50 base pair of the SNP within the PCR amplicon; and
4) (Optionally) The minor allele frequency is greater than 0.4 (sometimes across a range of populations).
Examples of fetal identifiers are set forth in Table 16. In some embodiments, the method of detecting the presence or absence of fetal nucleic acid in a sample comprises obtaining or possessing a nucleic acid sample known to be of maternal origin and suspected of comprising fetal nucleic acid; analyzing the nucleic acid sample to determine the maternal genotype at one or more nucleotide polymorphisms selected from the group consisting of the polymorphisms set forth in Table 16; and analyzing the nucleic acid sample to determine the fetal genotype of one or more nucleotide polymorphisms selected from the group consisting of the polymorphisms set forth in Table 16, where a fetal genotype possessing a paternally-inherited allele indicates the presence of fetal nucleic acid, further where nucleic acid comprising a maternal allele is digested using methods provided herein. In some embodiments, one or more of the polymorphisms set forth in Table 16 are used in conjunction with methods provided herein. In another some embodiments, one or more of the multiplex schemes provided in Table 11 is used according to methods provided herein. In certain embodiments, the maternal genotypes are first determined from DNA that is substantially free of fetal nucleic acid. For example, where the sample is blood of from blood, the maternal genotypes may be determined from the portion of the blood that comprises nucleated maternal cells (e.g., white blood cells). In some embodiments, the DNA that is substantially free of fetal nucleic acid is from peripheral blood mononuclear cells. In certain embodiments, the amount of fetal DNA is determined by comparing the relative amount of paternally-inherited alleles to an internal control (e.g., competitor oligonucleotide).
In Table 11, each primer of the amplification primer pair may comprise the entire sequence shown or only the non-underlined sequence, where the underlined portion of the primer is a tag sequence (ACGTTGGATG) (SEQ ID NO: 1) for improved multiplexing and the non-underlined portion is a sequence-specific primer sequence. The tag sequence may be any tag sequence known in the art that improves multiplexing. In certain embodiments, the invention in part includes primers that are substantially similar to the primers provided herein, for example, about 90% or more identical (e.g., primers differ by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotide mismatches, or 1-3 nucleotide mismatches, when aligned with one another), and further where the primers are still specific for a given nucleic acid region. For example, one or more bases of a primer sequence may be changed or substituted, for example with an inosine, but the primer still maintains the same specificity and plexing ability. Bases indicated by uppercase text are complementary to the nucleic acid sequence to which the primer hybridizes, and bases indicated by lowercase text are not complementary to the nucleic acid sequence to which the primer hybridizes. Bases indicated in lower case text can be selected to shift or adjust the mass of primers and amplification products.
In particular embodiments, a sequence tag is attached to a plurality of primary and secondary primer pairs provided in Table 11. The sequence tag can be attached to either one or both of the primary and secondary primers from each pair. Typically, the sequence tag is attached to the primary and secondary primer of each pair. The sequence tags used herein can range from 5 up to 20, from 5 up to 30, from 5 up to 40, or from 5 up to 50 nucleotides in length, with a sequence tag of 10-mer length being particularly useful in methods provided herein. The sequence tag need not be the same sequence for each primer pair in the multiplexed amplification reaction, nor the same sequence for a primary and secondary primer within a particular amplification pair. In a particular embodiment, the sequence tag is the same for each primer in the multiplexed amplification reaction. For example, in certain embodiments, the sequence tag is a 10-mer, such as -ACGTTGGATG- (SEQ ID NO: 1), and is attached to the 5′ end of each primary and secondary primer. In particular embodiments of methods provided herein, only a single primer pair is used to amplify each particular nucleic acid target-region.
In certain embodiments, methods provided herein may be used to improve the detection the Y-chromosome in a maternal sample, which may be used to determine the sex of a fetus. The presence or absence of the Y-chromosome in a maternal sample may be determined by performing the SRY assay described in Example 3. The SRY assay is a highly sensitive quantitative internal standard assay that detects trace amounts of the Y-chromosome. In certain embodiments, other polymorphisms located on the Y-chromosome may be assayed according to methods provided herein.
The presence or absence of the Y-chromosome in a maternal sample may also be determined by performing the AMG assay provided herein. The presence or absence of a target nucleic acid may be determined in combination with other assays, such as an RhD assay, blood type assay or sex test assay. Methods provided herein may also be used for other applications, including but not limited to, paternity testing, forensics or quality control assays.
In addition to prenatal applications, methods provided herein find utility in a range of applications, including, but not limited to, detecting rare cancer mutations, detecting transplant rejection and forensics.
In certain embodiments, the total copy number of nucleic acid molecules for the human serum albumin (ALB) gene is determined. Methods for determining the total copy number of nucleic acid present in a sample comprise detecting albumin-specific extension products and comparing the relative amount of the extension products to competitors introduced to the sample. In certain embodiments, the invention in part provides compositions and methods to determine the relative amount of fetal DNA in a sample (e.g., when the sample is plasma from a pregnant woman carrying a male fetus), which comprises annealing one or more albumin gene sequences to the fetal DNA, the primers provided in
Methods described herein provide a number of advantages. Methods provided herein allow a high sensitivity to detect polymorphic alleles (e.g., fetal identifiers) present at low relative percentages in a DNA mixture and present at low copy number, for example. Methods provided herein may also be incorporated into multiplexed assays in a single reaction in certain embodiments. Methods described herein are readily implemented, and only add a single additional step to the many current detection methods, for example.
Nucleases
Cleavage methods and procedures for selecting restriction enzymes for cutting nucleic acid at specific sites are well known to the skilled artisan. For example, many suppliers of restriction enzymes provide information on conditions and types of DNA sequences cut by specific restriction enzymes, including New England BioLabs, Pro-Mega Biochems, Boehringer-Mannheim, and the like. Nucleic acid to be cleaved often is/are free of certain contaminants such as phenol, chloroform, alcohol, EDTA, detergents, or excessive salts, all of which can interfere with restriction enzyme activity, in certain embodiments.
Embodiments of the invention can be assembled from multiple restriction endonucleases (available from various vendors) that are chosen to correspond to appropriate polymorphic alleles, as long as a restriction endonuclease selects for one polymorphic allele over another and performs a digestion within the amplicon sequence such that it prevents a subsequent amplification event. In some embodiments, the amplicon is chosen such that it contains a variable nuclease restriction site and sequence identifier, which may or may not be the same as the restriction site. Also, the restriction enzyme need not cleave at the polymorphic site, for example, at the variable nucleotide of a SNP.
Restriction enzymes are traditionally classified into three types on the basis of subunit composition, cleavage position, sequence-specificity and cofactor-requirements. However, amino acid sequencing has uncovered extraordinary variety among restriction enzymes and revealed that at the molecular level there are many more than three different kinds.
Type I enzymes are complex, multisubunit, combination restriction-and-modification enzymes that cut DNA at random far from their recognition sequences. Originally thought to be rare, we now know from the analysis of sequenced genomes that they are common. Type I enzymes are of considerable biochemical interest but they have little practical value since they do not produce discrete restriction fragments or distinct gel-banding patterns.
Type II enzymes cut DNA at defined positions close to or within their recognition sequences. They produce discrete restriction fragments and distinct gel banding patterns, and they are the only class used in the laboratory for DNA analysis and gene cloning. Type II enzymes frequently differ so utterly in amino acid sequence from one another, and indeed from every other known protein, that they likely arose independently in the course of evolution rather than diverging from common ancestors.
The most common type II enzymes are those like HhaI, HindIII and NotI that cleave DNA within their recognition sequences. Enzymes of this kind are available commercially. Most recognize DNA sequences that are symmetric because they bind to DNA as homodimers, but a few, (e.g., BbvCI: CCTCAGC) recognize asymmetric DNA sequences because they bind as heterodimers. Some enzymes recognize continuous sequences (e.g., EcoRI: GAATTC) in which the two half-sites of the recognition sequence are adjacent, while others recognize discontinuous sequences (e.g., BgII: GCCNNNNNGGC (SEQ ID NO: 2)) in which the half-sites are separated. Cleavage leaves a 3′-hydroxyl on one side of each cut and a 5′-phosphate on the other. They require only magnesium for activity and the corresponding modification enzymes require only S-adenosylmethionine. They tend to be small, with subunits in the 200-350 amino acid range.
The next most common type II enzymes, usually referred to as ‘type IIs’ are those like FokI and AlwI that cleave outside of their recognition sequence to one side. These enzymes are intermediate in size, 400-650 amino acids in length, and they recognize sequences that are continuous and asymmetric. They comprise two distinct domains, one for DNA binding, the other for DNA cleavage. They are thought to bind to DNA as monomers for the most part, but to cleave DNA cooperatively, through dimerization of the cleavage domains of adjacent enzyme molecules. For this reason, some type IIs enzymes are much more active on DNA molecules that contain multiple recognition sites. A wide variety of Type IIS restriction enzymes are known and such enzymes have been isolated from bacteria, phage, archeabacteria and viruses of eukaryotic algae and are commercially available (Promega, Madison Wis.; New England Biolabs, Beverly, Mass.). Examples of Type IIS restriction enzymes that may be used with methods described herein include, but are not limited to enzymes such as those listed in Table IA.
A third major kind of type II enzyme, more properly referred to as “type IV” are large, combination restriction-and-modification enzymes, 850-1250 amino acids in length, in which the two enzymatic activities reside in the same protein chain. These enzymes cleave outside of their recognition sequences; those that recognize continuous sequences (e.g., Eco57I: CTGAAG) cleave on just one side; those that recognize discontinuous sequences (e.g., BcgI: CGANNNNNNTGC (SEQ ID NO: 3)) cleave on both sides releasing a small fragment containing the recognition sequence. The amino acid sequences of these enzymes are varied but their organization are consistent. They comprise an N-terminal DNA-cleavage domain joined to a DNA-modification domain and one or two DNA sequence-specificity domains forming the C-terminus, or present as a separate subunit. When these enzymes bind to their substrates, they switch into either restriction mode to cleave the DNA, or modification mode to methylate it.
As discussed above, the length of restriction recognition sites varies. For example, the enzymes EcoRI, SacI and SstI each recognize a 6 base-pair (bp) sequence of DNA, whereas NotI recognizes a sequence 8 bp in length, and the recognition site for Sau3AI is only 4 bp in length. Length of the recognition sequence dictates how frequently the enzyme will cut in a random sequence of DNA. Enzymes with a 6 bp recognition site will cut, on average, every 46 or 4096 bp; a 4 bp recognition site will occur roughly every 256 bp.
Different restriction enzymes can have the same recognition site—such enzymes are called isoschizomers. Table IB shows that the recognition sites for SacI and SstI are identical. In some cases isoschizomers cut identically within their recognition site, but sometimes they do not. Isoschizomers often have different optimum reaction conditions, stabilities and costs, which may influence the decision of which to use. Table IB is provided only to show exemplary restriction enzymes, and does not limit the scope of the invention in any way.
Restriction recognition sites can be unambiguous or ambiguous. The enzyme BamHI recognizes the sequence GGATCC and no others; therefore it is considered “unambiguous.” In contrast, HinfI recognizes a 5 bp sequence starting with GA, ending in TC, and having any base between (in Table IB, “N” stands for any nucleotide). HinfI has an ambiguous recognition site. XhoII also has an ambiguous recognition site: Py stands for pyrimidine (T or C) and Pu for purine (A or G), so XhoII will recognize and cut sequences of AGATCT, AGATCC, GGATCT and GGATCC.
The recognition site for one enzyme may contain the restriction site for another. For example, note that a BamHI recognition site contains the recognition site for Sau3AI. Consequently, all BamHI sites will cut with Sau3AI. Similarly, one of the four possible XhoII sites will also be a recognition site for BamHI and all four will cut with Sau3AI.
Also from Table IB, most recognition sequences are palindromes—they read the same forward (5′ to 3′ on the top strand) and backward (5′ to 3′ on the bottom strand). Most, but certainly not all recognition sites for commonly-used restriction enzymes are palindromes. Most restriction enzymes bind to their recognition site as dimers (pairs).
Nucleic Acid Detection
Whether detecting sequence differences, detecting amplification products or primer extension products, any detection or discrimination method known may be utilized. These methods include, but are not limited to, primer extension reactions, mass spectrometry, hybridization using at least one probe, hybridization using at least one fluorescently labeled probe, direct sequencing, cloning and sequencing, and electrophoresis. Polymorphism detection methods known may also include, for example, microsequencing methods, ligase sequence determination methods (e.g., U.S. Pat. Nos. 5,679,524 and 5,952,174, and WO 01/27326), digital PCR (U.S. Pat. No. 6,143,496), mismatch sequence determination methods (e.g., U.S. Pat. Nos. 5,851,770; 5,958,692; 6,110,684; and 6,183,958), microarray sequence determination methods, restriction fragment length polymorphism (RFLP) procedures, PCR-based assays (e.g., TAQMAN® PCR System (Applied Biosystems)), nucleotide sequencing methods, hybridization methods, conventional dot blot analyses, single strand conformational polymorphism analysis (SSCP, e.g., U.S. Pat. Nos. 5,891,625 and 6,013,499; Orita et al., Proc. Natl. Acad. Sci. U.S.A 86: 27776-2770 (1989)), denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis, mismatch cleavage detection, and techniques described in Sheffield et al., Proc. Natl. Acad. Sci. USA 49: 699-706 (1991), White et al., Genomics 12: 301-306 (1992), Grompe et al., Proc. Natl. Acad. Sci. USA 86: 5855-5892 (1989), and Grompe, Nature Genetics 5: 111-117 (1993), detection by mass spectrometry (e.g., US 20050079521, which is hereby incorporated by reference), real time-PCR (e.g., U.S. Pat. Nos. 5,210,015, 5,487,972, both of which are hereby incorporated by reference), or hybridization with a suitable nucleic acid primer specific for the sequence to be detected. Suitable nucleic acid primers can be provided in a format such as a gene chip.
Primer extension polymorphism detection methods, also referred to herein as “microsequencing” methods, typically are carried out by hybridizing a complementary oligonucleotide to a nucleic acid carrying the polymorphic site. In these methods, the oligonucleotide typically hybridizes adjacent to the polymorphic site. As used herein, the term “adjacent” refers to the 3′ end of the extension oligonucleotide being sometimes 1 nucleotide from the 5′ end of the polymorphic site, often 2 or 3, and at times 4, 5, 6, 7, 8, 9, or 10 nucleotides from the 5′ end of the polymorphic site, in the nucleic acid when the extension oligonucleotide is hybridized to the nucleic acid. The extension oligonucleotide then is extended by one or more nucleotides, often 1, 2, or 3 nucleotides, and the number and/or type of nucleotides that are added to the extension oligonucleotide determine which polymorphic variant or variants are present. Oligonucleotide extension methods are disclosed, for example, in U.S. Pat. Nos. 4,656,127; 4,851,331; 5,679,524; 5,834,189; 5,876,934; 5,908,755; 5,912,118; 5,976,802; 5,981,186; 6,004,744; 6,013,431; 6,017,702; 6,046,005; 6,087,095; 6,210,891; and WO 01/20039. The extension products can be detected in any manner, such as by fluorescence methods (see, e.g., Chen & Kwok, Nucleic Acids Research 25: 347-353 (1997) and Chen et al., Proc. Natl. Acad. Sci. USA 94/20: 10756-10761 (1997)) and by mass spectrometric methods (e.g., MALDI-TOF mass spectrometry). Oligonucleotide extension methods using mass spectrometry are described, for example, in U.S. Pat. Nos. 5,547,835; 5,605,798; 5,691,141; 5,849,542; 5,869,242; 5,928,906; 6,043,031; 6,194,144; and 6,258,538.
Microsequencing detection methods often incorporate an amplification process that precedes the extension step. The amplification process typically amplifies a region from a nucleic acid sample that comprises the polymorphic site. Amplification can be carried out by utilizing a pair of oligonucleotide primers in a polymerase chain reaction (PCR), in which one oligonucleotide primer typically is complementary to a region 3′ of the polymorphism and the other typically is complementary to a region 5′ of the polymorphism. A PCR primer pair may be used in methods disclosed in U.S. Pat. Nos. 4,683,195; 4,683,202, 4,965,188; 5,656,493; 5,998,143; 6,140,054; WO 01/27327; and WO 01/27329 for example. PCR primer pairs may also be used in any commercially available machines that perform PCR, such as any of the GENEAMP® Systems available from Applied Biosystems, for example.
A microarray can be utilized for determining whether a polymorphic variant is present or absent in a nucleic acid sample. A microarray may include any oligonucleotides described herein, and methods for making and using oligonucleotide microarrays suitable for prognostic use are disclosed in U.S. Pat. Nos. 5,492,806; 5,525,464; 5,589,330; 5,695,940; 5,849,483; 6,018,041; 6,045,996; 6,136,541; 6,142,681; 6,156,501; 6,197,506; 6,223,127; 6,225,625; 6,229,911; 6,239,273; WO 00/52625; WO 01/25485; and WO 01/29259, for example. A microarray typically comprises a solid support and the oligonucleotides may be linked to this solid support by covalent bonds or by non-covalent interactions. The oligonucleotides may also be linked to the solid support directly or by a spacer molecule. A microarray may comprise one or more oligonucleotides complementary to a polymorphic site within a nucleotide sequence
The Examples hereafter illustrate embodiments of the invention and are not limiting.
The effectiveness of restriction endonuclease enhanced polymorphic sequence detection was demonstrated using several restriction endonucleases (REs), including HpyCH4V and NlaIII (purchased from New England BioLabs, Inc). Both of these enzymes were separately tested in multiplexed genotyping reactions for their ability to specifically cleave one allele of a given polymorphism while allowing PCR amplification of the remaining allele of the polymorphism. See Table 2 for the polymorphisms tested with each enzyme.
Two CEPH DNA samples were mixed in varying ratios to generate DNA samples composed of 0%, 2%, 5%, 20%, 50% and 100% DNA heterozygous for both alleles of the SNP, with the remaining DNA being homozygous for the allele recognized by the RE. Table 3 shows DNA samples used in these studies and corresponding genotype information. Mixtures composed of NA05995 and NA10849 were used for experiments with HpyCH4V enzyme, and mixtures composed of NA10862 and NA10846 were used for experiments with NlaIII enzyme.
After preparation of the sample DNA mixtures, PCR cocktail was prepared according to Table 5 below (using multiplexed PCR primers as shown in Table 6) to either include no restriction endonuclease or 0.25U of restriction endonuclease per each sample reaction. PCR cocktail was aliquoted to a 96-well plate to include 7 replicates of each DNA mixture for each enzyme condition. After addition of DNA to the PCR cocktail mixtures, samples were incubated at 37° C. for 1 hour to allow enzyme digestion of DNA samples and then immediately thermal cycled using standard conditions (Table 7).
ACGTTGGATGCACAAGATTCTGAAACTTAG
ACGTTGGATGGCTGTTTAACTCAGCATG
ACGTTGGATGTTGGGTGCAGAGTAGTCATC
ACGTTGGATGTTCTAGCTTGCTTCTCCTCC
ACGTTGGATGATGTCCACCTCCTGCTCCAC
ACGTTGGATGGAAAGTTGTCGTGGTAGAGG
ACGTTGGATGCTAGCGTACCCAATGGAATC
ACGTTGGATGCTAACCAGGAAAAGACACCC
ACGTTGGATGGTGGTAGAAACAAATGTCAGC
ACGTTGGATGCTGCTAAGCATGAGAGAAAG
ACGTTGGATGGGCCTGTTCATTCTCAGAAA
ACGTTGGATGTGACTAGGAAATCACACTGG
ACGTTGGATGTAGCCTTTAGTCTTGATGCC
ACGTTGGATGCCATTCTTGTATGTTTTGTC
ACGTTGGATGGCCTCAGTAGTCACATAAGG
ACGTTGGATGTTGAGATCAGTGTCGGTTCC
Amplicons generated during PCR were genotyped with the extend primers in Table 5 using standard iPLEX™ assay and MassARRAY® technology (Jurinke, C., Oeth, P., van den Boom, D., MALDI-TOF mass spectrometry: a versatile tool for high-performance DNA analysis. Mol. Biotechnol. 26, 147-164 (2004); and Oeth, P. et al., iPLEX™ Assay: Increased Plexing Efficiency and Flexibility for MassARRAY® System through single base primer extension with mass-modified Terminators. SEQUENOM Application Note (2005), both of which are hereby incorporated by reference).
Results
Digestion of DNA with both restriction enzymes allowed detection of minor alleles when they were present at ratios as low as 2% heterozygous DNA. This is in contrast to undigested DNA samples where minor alleles were only reliably detected when present at ratios of 20% heterozygous DNA and higher. When allele peak area ratios are considered, the effect of restriction endonuclease digest is even more apparent. HpyCH4V digested samples showed minor allele peak area ratios of 0.35-0.45 in 2% heterozygous DNA mixtures, while minor allele peak area ratios of 2% heterozygous DNA mixtures were at background levels without enzyme digestion (
Optimization Studies
Initial optimization studies for enzyme concentration and pre-PCR incubation time of HpyCH4V digestion were performed using 5% heterozygous DNA mixtures (0.6 ng heterozygous DNA, 11.4 ng homozygous DNA). Based on these experiments, maximal peak area ratios were obtained with incubation times as short as 5 minutes and 0.25U HpyCH4V enzyme.
A similar experiment was performed as described in Example 1 using a different restriction endonuclease, TfiI. In this experiment, the Tfil restriction endonuclease selectively recognized and cleaved the ‘C’ allele of the ‘C/T’ SNP, rs4487973. The SNP rs4487973 occurs in the following genomic sequence on chromosome 1: CACACAGTTAGGATT[C/T]ACCTGAGCTTGTCCC (SEQ ID NO: 28). For these studies, two CEPH DNA samples, one homozygous ‘C’ and the other heterozygous ‘C/T’ for the rs4487973 SNP, were mixed in varying ratios to generate DNA mixtures containing 0%, 1%, 2.5%, 10%, 50% of the rs4487973 ‘T’ allele. The TfiI restriction endonuclease was either added or not added to each mixture to determine the endonucleases' effect on detecting the polymorphic sequence. Of the mixtures not digested with Tfil enzyme, the rs4487973 ‘T’ allele was detected in the 10%, and 50% ‘T’ allele mixtures, but not the 0%, 1%, and 5% ‘T’ allele DNA mixtures. However, of samples digested with Tfil enzyme, the rs4487973 ‘T’ allele was detectable in 1%, 5%, 10% and 50% ‘T’ allele mixtures. These results indicate the utility of this method to improve detection of polymorphic alleles present at low relative concentrations in a sample.
Selection of SNPs
Analysis of paternally-inherited alleles in clinical samples and correlation with Y-chromosome frequency in male fetuses was performed with a total of 16 SNPs. SNP assays for analysis of clinical samples were multiplexed as 8-plexes. All SNPs had a minor allele frequency (maf) of ˜0.4 in all ethnic groups and were unlinked.
For performance evaluation of a universal Fetal Identifier panel that can be multiplexed with disease-specific markers, a new panel of 87 NT SNPs with a pan-ethnic maf>0.4 was selected and multiplexed into 16-plexes.
Method of SNP Analysis
Analysis of SNPs in maternal buffy coat and maternal plasma was performed using the iPLEX™ assay and MassARRAY® technology. In brief, the target region surrounding the SNP is first amplified by PCR. Subsequently an oligonucleotide primer is annealed to the PCR product and is extended allele-specifically by a single nucleotide using a mixture of 4 terminator nucleotides and a DNA polymerase. The extension products are transferred to a miniaturized chip array and are analyzed by MALDI-TOF Mass Spectrometry. Determination of the molecular mass of extension products allows unambiguous identification of the SNP allele present in the sample. The peak area ratio of mass signals allows the estimation of the relative abundance of the alleles in a given sample.
Clinical Samples
The total sample set consisted of 35 paired blood/plasma samples from pregnant Caucasian woman (nine 1st trimester; twelve 2nd trimester; fourteen 3rd trimester). The subset of samples used for correlation of Y-chromosome frequency and paternally-inherited alleles in maternal plasma consisted of 19 samples of pregnant Caucasian woman carrying a male fetus.
DNA Extraction
DNA extraction was performed from 1 ml of maternal plasma using the Qiagen® MinElute kit for fetal genotyping. DNA extraction from frozen blood (minus plasma) was performed from 4 ml using Qiagen's PureGene kit for maternal genotyping.
Results
An assay targeting sequence differences in the Amelogenin region on the X and Y chromosome was used to assess the relative amount of fetal DNA extracted from plasma of pregnant woman carrying a male fetus. Details of the AMG assay are depicted in
Sixteen of nineteen (84%) plasma samples with a male fetus showed a Y-chromosome frequency of higher than 5%, indicating presence of at least 10% fetal DNA in the extracted DNA.
Table 8 provides a list of SNPs that were multiplexed at 10+ plexing level and passed all phases of the validation. The following shows the validation scheme, performance criteria and model system used to qualify multiplex SNP assays for their utility in identifying the presence for fetal DNA.:
Phase I
Multiplexed SNPs passing Phase I test criteria are tested in Phase II
Phase II
Multiplexed SNPs passing Phase II test criteria are tested in Phase III
Phase III
Application of this assay panel to a model system for the detection of fetal DNA in maternal background showed that paternally-inherited fetal alleles can be detected with a sensitivity of 95% at 100% specificity if the sample preparation method can enrich the relative amount of fetal DNA to 20%. In Table 8, the minor allele frequency (MAF) for each SNP from different ethnic populations is provided. The ethnic populations are defined by the HapMap Project, where CEU represents individuals of Northern and Western Europe descent, HCB represents Han Chinese in Beijing, JAP represents Japanese in Tokyo, and YRI represents the Yoruba in Ibadan, Nigeria.
A multiplexed panel of 16 SNPs was analyzed with maf>0.3 in the same maternal plasma DNA extraction and established a baseline of maternal genotypes by analyzing DNA from PBMCs. Using the maternal genotype information, paternally-inherited alleles were identified in plasma samples and estimated the amount of fetal DNA from the peak area ratio of extension products representing paternally-inherited fetal alleles and maternal alleles.
The AMG XY frequency was then compared with the allele-frequency of paternally-inherited fetal alleles in informative SNPs. This comparison revealed that samples with a positive Y-frequency of 10% (used as a Limit-of-quantitation threshold) or more have significantly higher differences between maternally and paternally-inherited fetal allele-frequencies (p-value<0.001; Fishers' exact test). This data suggests that Fetal Identifiers can be used as a non-gender specific approach for identification of the presence of fetal DNA.
The effectiveness of restriction endonuclease enhanced polymorphic sequence detection was demonstrated using Tsp509I (purchased from New England BioLabs, Inc). Tsp509I was tested in multiplexed genotyping reactions for its ability to specifically cleave one allele of a given polymorphism while allowing PCR amplification of the remaining allele of the polymorphism. See Table 9 for Tsp509I enzyme characteristics.
E. coli expressing cloned Tsp509I gene
Potential SNPs for use with Tsp509I
SNPs meeting the allele frequency criteria above were further screened for three characteristics:
Multiplexing Tsp509I SNPs
Multiplexed assays were designed using 274 SNPs from Table 10. The resulting multiplexed SNPs are shown in Table 11A with associated PCR primers and Extend primer for each SNP, and genomic sequence comprising the amplicon sequence (with the SNP allele variants indicated by brackets) are shown in Table 11B.
ACGTTGGATGTGTCAGACTTGTCTGAAGGC
ACGTTGGATGCAGATAGTGCTTGAGAGGAG
ACGTTGGATGCCAAAGTGAACTTGGGTCTC
ACGTTGGATGGGGAGAAAGAAACAACCTGC
ACGTTGGATGAAGCCTGTGGACTGTTAACC
ACGTTGGATGGACATTAAGCCCAAAACAGG
ACGTTGGATGGTGAACTTTTTTTGCAAGGG
ACGTTGGATGAAAGCTGGCCAGGGATATAG
ACGTTGGATGCATCAGCAGTGTGTAAGAGG
ACGTTGGATGCTGGTGAGTAAGCATTGAAG
ACGTTGGATGTCCAAGGTGGTCTTTTGGAG
ACGTTGGATGGTTGGTAAATGGTAGAGCCG
ACGTTGGATGTTTGTTAGCAGCTATGCTGG
ACGTTGGATGGGAGTAGTCTTCACCTGTAG
ACGTTGGATGGTCTCTTAAGCAACGAGCGG
ACGTTGGATGAGAAGGGCAACCAACAACTG
ACGTTGGATGACACTGTTCGCATCTGCATC
ACGTTGGATGGTAGCTCAGGCAAGGAGATT
ACGTTGGATGTGTTTCTCAGGAGTTCCCAG
ACGTTGGATGGCAGGAGAGGAGAAAAAGAC
ACGTTGGATGTGTGTCCAGTGACCATAAGG
ACGTTGGATGGTTGACGCAAAGCAAGTGAC
ACGTTGGATGTGGTTCCAGTTCTCAAGCTC
ACGTTGGATGTCTTAGGAAACCACGTCCAC
ACGTTGGATGATTGTGGCTGTGCTGTCCTC
ACGTTGGATGCGTATCTGTCTTGGATCCTG
ACGTTGGATGGGAACACCTCCATTCTGATG
ACGTTGGATGACACAAGTGGGAGAGGTTTG
ACGTTGGATGACACCATCTCGGTAGGAAAG
ACGTTGGATGCAGTTTGTTAGGTTCTCTGG
ACGTTGGATGCAGGAAGTATATGAGATCTGG
ACGTTGGATGTACACAGTAAGTTCCCTGAG
ACGTTGGATGAAGGGTGAGGTGAGATAACG
ACGTTGGATGCTGGTTCCAGCACAAGTTTC
ACGTTGGATGGAGTGAGTCCTTTGATCCAG
ACGTTGGATGCTACTTCCAAAGATTGTTG
ACGTTGGATGCTCACAGTGAAAGTGAACAG
ACGTTGGATGCCCGTATATGTAGCCACTTT
ACGTTGGATGGACATATAATACCTTGGTCCC
ACGTTGGATGCGTCTGCTTCCTTCATAGAG
ACGTTGGATGGAGAGGTTGGGAAAAATGTG
ACGTTGGATGGGAAGATGCACCACTTTCTG
ACGTTGGATGAGCTTTCCTAAACCTGTGAC
ACGTTGGATGAAACCACTTCCTGCTTTCCG
ACGTTGGATGACCACCATCACAAAAAGAGG
ACGTTGGATGTATGTTTGCATGTTGTTTG
ACGTTGGATGCCATGTGAGGAGGCATGTTT
ACGTTGGATGTTAATGCCAGACAAGCCTCC
ACGTTGGATGGTATTGGGTTACATGATG
ACGTTGGATGAGAAGGTCCTGTTAGTAGGG
ACGTTGGATGGCGTGCATGGACTTCACAAG
ACGTTGGATGCCACTGGCCTTTTCAAAGTC
ACGTTGGATGGAGCATCTTCAAATATCCCC
ACGTTGGATGAACAACCGTTTTCTCTTGGG
ACGTTGGATGAATCCATTTCAGACGCAGCC
ACGTTGGATGAATGTCAGAGATCACAAGCC
ACGTTGGATGGGAACTGATGGAAGAAAAGC
ACGTTGGATGCCTTTTGTGAGCAAGATGCC
ACGTTGGATGTGGGCACTGTAATACAAAGG
ACGTTGGATGTCCACACATGGTATCACAAC
ACGTTGGATGCTGTTGCCTAAAGTTCTCGC
ACGTTGGATGACATTACTTGAGACCCACAC
ACGTTGGATGGTGATATTGAGTCTCACCTG
ACGTTGGATGCTCTTTCTCATTATCATTC
ACGTTGGATGTGGTCCAGTAGGAAAACAGG
ACGTTGGATGTTCACTGACTCATGGATGGG
ACGTTGGATGGCATTTTGGGAAATAATACC
ACGTTGGATGGGGAAGTCAGGATGAAAGTG
ACGTTGGATGTACCTTCTATATCCAAGGAC
ACGTTGGATGATCCTCCCTTTTGAAACTTG
ACGTTGGATGTCAGAAGGAGAAGTACCAGC
ACGTTGGATGGCTAGGATTACACGTGTGAG
ACGTTGGATGCTGTCTGTGTGATCATCAGG
ACGTTGGATGTGACGCTAAAGACTGAGTGG
ACGTTGGATGCATTGAAACCTGGGATACAC
ACGTTGGATGAAAGGCAATCTCGACCTCAC
ACGTTGGATGTGCTCACACAAAGCCTGTTG
ACGTTGGATGGAATAACTATGAGCTCATGG
ACGTTGGATGTGAATCCCATGAGCATGAGC
ACGTTGGATGATTCCACACAGCATTGCCTC
ACGTTGGATGGCCTTATTAGCTCTCACTTG
ACGTTGGATGCATCTCATGAGAAAGGCATC
ACGTTGGATGGAAAGGCCACAAAGCTGTTG
ACGTTGGATGCACATGCATGAGTATGGGAC
ACGTTGGATGTGCAGAGCTGCGAGAAGAAG
ACGTTGGATGAAGAGAAGGGCTTTGCATCC
ACGTTGGATGAGCAAGTGTTCCCTTTTTGG
ACGTTGGATGCACGCGTAGGCTATGGTTTA
ACGTTGGATGAAGTGCTGGGATTACAGGAG
ACGTTGGATGGAGACAGGCAAAGATGCAAC
ACGTTGGATGGAGACGATTCTTCAGGAAAC
ACGTTGGATGCCATGACTCTAGTGACCTTC
ACGTTGGATGTAAGCATCCATGGACCTACC
ACGTTGGATGCAGGTGGTAAATGTGCTCAG
ACGTTGGATGTCTGAAGGTAGACCTGGATG
ACGTTGGATGCTCAGGATATCATTACACACC
ACGTTGGATGCGAGTTGAAGATCCCATACG
ACGTTGGATGCTCGGTGAACTATAGGAATC
ACGTTGGATGAAGCAACTGGCACTCCTAAG
ACGTTGGATGGAGTGTTGTGATGCATGCC
ACGTTGGATGTTATACAGGTTCCAGCCAGC
ACGTTGGATGCAGAGAGAAAAGGGAGTAGG
ACGTTGGATGTCTCAAATATCTAAGTGGG
ACGTTGGATGGCAAAACTTCACCTCAATAA
ACGTTGGATGCGGTTTCTTTTGAGGACTGG
ACGTTGGATGGCTCAGTGTCTGACAAAAGC
ACGTTGGATGACTGGCCATGCAGATGTAAG
ACGTTGGATGCACTGCCCATAGACTCTTTC
ACGTTGGATGGAGATGAGTAAGAGCAGGTG
ACGTTGGATGCTCATAAGACCCTGAACACC
ACGTTGGATGGAAAAATCCATCCTCTGAACC
ACGTTGGATGCCATGGCTCGTGTTCTTAAC
ACGTTGGATGGTCACTGAACTCTGGAGTAG
ACGTTGGATGGCAGTTTTCAAAGGAAACCC
ACGTTGGATGTCCCCCTACTTGCTTGAAAG
ACGTTGGATGTGAAAGAGTGAAGGGAGGAC
ACGTTGGATGCTCCCATCTATGATTTCCAG
ACGTTGGATGATGCATATCTGGAGACACAC
ACGTTGGATGAATGAGAGCTTGCTTACTTC
ACGTTGGATGAGTGTCGTTCAGACACTAGC
ACGTTGGATGCATTGCAGTAACTGGAGGTC
ACGTTGGATGGGCACAGTAGTTCAGTTACC
ACGTTGGATGAACACCAAGGAAAGCGGATG
ACGTTGGATGCTGCTTAGTAACTTCTGTCC
ACGTTGGATGTAATACCCTGAGCAAGGACG
ACGTTGGATGGTGCATTTAAAATCCATGTG
ACGTTGGATGGTCCATTTAACGGTGTGGAG
ACGTTGGATGGGTTCATGAAATGTTAGTTCC
ACGTTGGATGCACCAGTGCAAACACACAAC
ACGTTGGATGCCTGATTGTTTTGGAAGGAG
ACGTTGGATGTGTGCAGCACTTTTCACAAG
ACGTTGGATGCAGGGTCACATCACAGATTG
ACGTTGGATGGGATCAAGAGGAAAAAATGGG
ACGTTGGATGTAGTTTCAATCTCTGTGCTG
ACGTTGGATGTACATTCAGACGATAGTGCC
ACGTTGGATGAGACCAAGTAACCCCAAACC
ACGTTGGATGCACATGCTAGAGAAAGAGGG
ACGTTGGATGTATGTCCTTCCCTGATTTTC
ACGTTGGATGAGTTGCCATGTTTCCACAGG
ACGTTGGATGGACTAATACTCAGGTTGAGG
ACGTTGGATGCTACGTGACCCAAAGTTCAG
ACGTTGGATGTCTCACTCCTGGTTACCTAC
ACGTTGGATGGGGCTCTTATTATTGTACTC
ACGTTGGATGAACAAGCCCAAGTTCTCCAG
ACGTTGGATGAGAAGGAGGTCATTCTAGGC
ACGTTGGATGACATGGACTCTAAAGCCACC
ACGTTGGATGGGCAGAACAAGGACAGATAG
ACGTTGGATGAGTCTAGTAAAAGTTCTGCC
ACGTTGGATGGCCAGCTTGTCCATTAAAGG
ACGTTGGATGCTGGCTTATAAATAAAAGACC
ACGTTGGATGTTTCACAGGGTTAGGATGGG
ACGTTGGATGCTAGCAAAGGCTGGATTCTG
ACGTTGGATGTGGAAGGCAGAGTGATATAC
ACGTTGGATGGCTTTCTTCACTCAGAAGGG
ACGTTGGATGGGGAAGGTGTTTGTCTCATA
ACGTTGGATGTGGTACAGTTTGAAAGGAGC
ACGTTGGATGAGAAGATATGTTGAGAGGGC
ACGTTGGATGTATTCCCTTTCTGGCTGTGG
ACGTTGGATGGTATGGTGCCTCCACAAAAG
ACGTTGGATGCCTCTGGATATATGTCCAGT
ACGTTGGATGATGGTGGTGGCAATATTGGG
ACGTTGGATGGCCAAAAAGCAGGCTTCTTC
ACGTTGGATGACCATTTATTGGCCCTGCTC
ACGTTGGATGATGGCAACATCTGCTTTCCC
ACGTTGGATGAGAATGACAAACCCAAGGGC
ACGTTGGATGGGACCTGTGCAAAACTTTGG
ACGTTGGATGGTAGGTTAAGAGGGAAAGGG
ACGTTGGATGACTTGCCTTGTTCTTGACTG
ACGTTGGATGAAAGTCAGCACAGTCACTGG
ACGTTGGATGTCTCGAACAAGCTAGAGGAC
ACGTTGGATGTCGAACTCCTGACCTCAAAC
ACGTTGGATGCCTGTAATCCCAGCACTTTG
ACGTTGGATGGTGTTCATACTGTAGGCTTG
ACGTTGGATGTAAACCAACCCCCTTCTTGC
ACGTTGGATGGTGTAATAGGCTTGTGAGAG
ACGTTGGATGCTCTTTGGATTAAATGCCTGC
ACGTTGGATGTGCTTCATAACTCTGTCACG
ACGTTGGATGCAAAACAGTATCGTAACAG
ACGTTGGATGCAAGTCTCCCTAGCTAAGTG
ACGTTGGATGTAGGAAGATCCTGGAAGGTG
ACGTTGGATGCATTTGCGGCAAAGAGGGAG
ACGTTGGATGAGCTCCCACACATGAAAGAG
ACGTTGGATGCACCCTATGCGACTTCTTTG
ACGTTGGATGGTGGAGCTGTTATTCTAGTG
ACGTTGGATGTATCCCCCAAACCTCACATC
ACGTTGGATGGAGTGGACTATAGTGGATGC
ACGTTGGATGATCATGGAAGTGATGAGAGG
ACGTTGGATGGCCACATTCAACTGCAGTTC
ACGTTGGATGAGGACCTGGAGCTCAGCAAC
ACGTTGGATGCTCAGCTGTCTCCATGCTC
ACGTTGGATGTGTGCAGCAATGATCACAG
ACGTTGGATGCTCAGCCATCTCCTGTCATC
ACGTTGGATGACAAGTAAGGTTGGGTGGTG
ACGTTGGATGCCTATTCATGGAACCTCCAC
ACGTTGGATGGGATGCTATATCATAGCCAC
ACGTTGGATGCTTCCCCCGCTCTTTTAAAC
ACGTTGGATGCTCTGCTCTGCACACATAAG
ACGTTGGATGCCCTGAGATTATGTGACACC
ACGTTGGATGGGTGTTAGTCAACTAGGAGG
ACGTTGGATGAGAGCTTGGACTCTAGCATC
ACGTTGGATGAGAGGAAGCAAAGCTAAGGG
ACGTTGGATGCCCAGACCACTTTATAAGCC
ACGTTGGATGAGGAGGTGACATTTAAGCTG
ACGTTGGATGCTTTGCACTTACTGCTTCCC
ACGTTGGATGACGGCTAATGCTCCTCATTC
ACGTTGGATGGCATGTTTAGTACCTGCAAG
ACGTTGGATGGGGCTTGAATAGCTAGATAC
ACGTTGGATGTTACCTAGCTAGAGATCTGG
ACGTTGGATGGATAGGGATAGACACAGGAC
ACGTTGGATGGTAGTTAAAGGTGAGCAGGG
ACGTTGGATGGGATTTCTGTGAAGCTGCTC
ACGTTGGATGAAAGTGTTGACCCCAGTGTG
ACGTTGGATGCCATGAATGGCAAGTGTCTG
ACGTTGGATGCTTGGGTTCTGAGGATTTGC
ACGTTGGATGTTGGTAGCATATGGGTCTCC
ACGTTGGATGCCTTTTCTGATGAATGAAGCC
ACGTTGGATGGCCAGGGATTGTATTCGAAG
ACGTTGGATGCTGGATATTGTTCAGCTGGG
ACGTTGGATGCTTCTATGAACCACCAAGGC
ACGTTGGATGGGATACAGCCAAACCATGTC
ACGTTGGATGGGTAGGAAACGTGTACACTG
ACGTTGGATGATCCATGAAAACAGGATGTC
ACGTTGGATGATGCCTATTTCTTGTGACCC
ACGTTGGATGCTATACTGCACCTTAGAACC
ACGTTGGATGATGACATACTCCCATGTGCC
ACGTTGGATGGAAGAAGAATCAGAGCCAGC
ACGTTGGATGCACGCTATGTAAAAGTAGCA
ACGTTGGATGCTTCCAAAGTTCATATGCAG
ACGTTGGATGGAATGGATAGAAGAATCTG
ACGTTGGATGACTACCCTGACTGCTATCTC
ACGTTGGATGCCTGAGTCAACCTTGGAAAG
ACGTTGGATGTAATAGCTCCCCCAACAGTC
ACGTTGGATGAGCCTGAATCTCTAGCAGTC
ACGTTGGATGGAGAGACACTGTCTCACTCA
ACGTTGGATGCTGCAAGCTAAGAAACACAC
ACGTTGGATGCGTACCATATACCTAGGGTG
ACGTTGGATGCCATTATTTCTCCCAAAGCTC
ACGTTGGATGAAATAAGACCCTTGCACCCG
ACGTTGGATGTTGTGGAAGGAGGCAAGGG
ACGTTGGATGATGTCTGTCTTGGCTATGGG
ACGTTGGATGAGAAGAGATGGTGGTTGTGC
ACGTTGGATGACTGTTAGCTAGCACTGTGG
ACGTTGGATGAGTATCCTCCAGTTTAAGGG
ACGTTGGATGGGACTCCCTACTCATTCAAG
ACGTTGGATGGTTGTTGCTAGTAGACCGAG
ACGTTGGATGCTAGTTTTCTCTTCCCCAGC
ACGTTGGATGTTTAGTGACACCTCCCATCC
ACGTTGGATGGGGTAGTAGGAAGTGGTTAG
ACGTTGGATGGGCATGCAACATAGACTTGG
ACGTTGGATGTTAGTGAGCATCAGAGGCAG
ACGTTGGATGAGGCTTTCAGGATCTGCTTC
ACGTTGGATGCTCAAGGGCCATAGAAACAC
ACGTTGGATGGTGGACACAGGACAGCATTG
ACGTTGGATGTCATCGCATCATGCATCCTC
ACGTTGGATGATGTGTCAAGACCATCTGGG
ACGTTGGATGACGGAGCAAGACTCTGTCTC
ACGTTGGATGAGTTATTCTCCCGAGAAGGC
ACGTTGGATGGCTAGGCAGATTGTGCTGTG
ACGTTGGATGGACATGGTTGTGTTGTGAAG
ACGTTGGATGAAGCACCGCTGGTGATAATG
ACGTTGGATGACTATGGGTAGTACATGGG
ACGTTGGATGGCATCATTTGAATATTCACAC
ACGTTGGATGTGCAAGCCCACAGGACAAAC
ACGTTGGATGTTCTTGTGGATTCCACTCCG
ACGTTGGATGGTTGGTAATAGCTACAGCCC
ACGTTGGATGAGAAGAGCTGACTGTCAGCG
ACGTTGGATGTTTTTGTCCCCAAACATCCC
ACGTTGGATGTTTAGTGAGGGTGCTGGAAG
ACGTTGGATGAGACATCAGAGAGAAGGGAC
ACGTTGGATGGTATTAAAGATGAGCCCACAG
ACGTTGGATGTTGTTCCTGACTTCAAGGGC
ACGTTGGATGACCAGTTCCTACCCATGAAG
ACGTTGGATGAAGGTGCTGTGGCAAGTTAG
ACGTTGGATGCTGCTGTGGGTATTCAGTTC
ACGTTGGATGTGGAGAAGAAACTCAAAAG
ACGTTGGATGACAGGGTCTGTACATTGCAG
ACGTTGGATGCCAGGTCTCAACACTGATTG
ACGTTGGATGGCCATTATGTGAAATCAGCG
ACGTTGGATGGAAATACTTCCCTCGGGCTC
ACGTTGGATGAACCGCAGGTAAGGATTCAG
ACGTTGGATGCTTCCCTGGCTTCATTTTCC
ACGTTGGATGCGATCTCCATCAAAAGAGGC
ACGTTGGATGAGAGATTGAGCTTCAGTCCC
ACGTTGGATGTCAGTCTTGTGTAGATAGGG
ACGTTGGATGCCACTTACAGAACAGAAGGG
ACGTTGGATGTATACCCATCCCCCAATGAC
ACGTTGGATGTGCTGCTGGATTCAGTTTGC
ACGTTGGATGGAACACTTTAGGCCAATATCC
ACGTTGGATGTTGATATGAGCCTCTGAGAC
ACGTTGGATGAGCTGAAGTTCGTGAGATCC
ACGTTGGATGGGACGTGAGCAAGAAAAGAC
ACGTTGGATGTGCTACGATTCAGTAATGAG
ACGTTGGATGGTGGTCTATTGAGGCAATGG
ACGTTGGATGAGGTTCATTTATGTGGTAGC
ACGTTGGATGGACCCATGTCTGTCATACTG
ACGTTGGATGTGGAGCACTTTTGATGTG
ACGTTGGATGTGTCAAAACCCCATCTCTAC
ACGTTGGATGGGGCTCAAGTGATTTTCCAG
ACGTTGGATGGCCAAGCAACACTATGGTAT
ACGTTGGATGAAGACCAAGTGAACTGTGCC
ACGTTGGATGAATGCCATTTCCTCAGGAGC
ACGTTGGATGGAAGCATCTAAGCACAGCTC
ACGTTGGATGGACTGGTAGAGTAAGTTCTG
ACGTTGGATGATTCCACATTCAGAGACAAC
ACGTTGGATGGTGTTGATCTGTCACATGGC
ACGTTGGATGGAGAACAAATAGCCCTGAAG
ACGTTGGATGGAGGTGCCAGCTAATCTAAC
ACGTTGGATGCATGAGGCCACAAAGGAAAG
ACGTTGGATGTATTACATCGAAATCAAGG
ACGTTGGATGAGGCAAAATCGTTTTCATCC
ACGTTGGATGGAAGTGTTTAGGATTTGAG
ACGTTGGATGTGCTCACTGGAGCATTTCAG
ACGTTGGATGAAGTCTTGACATAAGGTAG
ACGTTGGATGGGCAATCTTAAAGAGGGTTG
ACGTTGGATGCAAAAGCTTTGCGCATCAGG
ACGTTGGATGACAGGACCCTTGCTTTCAAC
ACGTTGGATGTTAGGCCAAGCTCATGCTTC
ACGTTGGATGAATCTGGCCAGGGAAGGTTG
ACGTTGGATGCAGTGGATTTCAAATCCGGC
ACGTTGGATGTGTTCAGAGGGTGTTGGATG
ACGTTGGATGCAATCAGCTACTGCTGATCC
ACGTTGGATGTGGTTTGGTTTCTCAGCTGG
ACGTTGGATGCTGTCAAAAGCCAGGCTAAG
ACGTTGGATGGAGGTTCAAAGAGTATAAAG
ACGTTGGATGGCAACCAGTTATCCCCATTC
ACGTTGGATGGTACTTTGTGACCTTGAGGC
ACGTTGGATGGCTTGCAGAGGTTCACTAAC
ACGTTGGATGTGAGGCCATTAAAAGCAGGG
ACGTTGGATGTAGAGCTCACAGAGCACTTC
ACGTTGGATGAGCACTTAACTGAGTCTGGG
ACGTTGGATGCTCAGTTTAAAGTCACTGCC
ACGTTGGATGTAACCCTGCAAAGACTAGAG
ACGTTGGATGTTTACAGACTAGCGTGACGG
ACGTTGGATGATCTCACGATCCCCCATTTC
ACGTTGGATGGGAGGATGAAATCAGTGGTC
ACGTTGGATGAGAACATGCCAGAAAGTGCC
ACGTTGGATGTTTTTCTGTCTCAGCCTCCC
ACGTTGGATGATGGAGAAACCTGTCTCTAC
ACGTTGGATGTATGGATGCAAGCCTTTCCC
ACGTTGGATGAGGCTGAAGAATGCTTTCCC
ACGTTGGATGAGCTTGGGCTGAATGTTAGG
ACGTTGGATGTAAAAGCAAAACAGCTTCCC
ACGTTGGATGGGCTCTAGTTTTCAGCAGAC
ACGTTGGATGCTCAAAACCTGGCTACCTTG
ACGTTGGATGGTTACACTGACAATCAAGGG
ACGTTGGATGACTCTCATGTACCCTCTCTG
ACGTTGGATGAACTGATGGCTCGTACTACC
ACGTTGGATGGCTCTTTTCCCTATGATGTG
ACGTTGGATGGATAATATTGTGCTGCATGCT
ACGTTGGATGACCTTGTTCTGTGTGTGTGG
ACGTTGGATGCTGGATCTTACCTCCATAGC
ACGTTGGATGACTAGAATCGTGCAGAGAAC
ACGTTGGATGGAGCACTTATCACAGGTCAG
ACGTTGGATGGAAGGTGGGATAAACAAGGG
ACGTTGGATGACTGAAGCATAACGCCTCTG
ACGTTGGATGGGTGCCCAAACATGTTATGC
ACGTTGGATGATCTTCATGTCCCAAGGAGG
ACGTTGGATGCCAAGTTTATGAAACGTAG
ACGTTGGATGTCACGTCAGACTACACTGAG
ACGTTGGATGGGATTATAGGCATGAGCCAC
ACGTTGGATGCATTGCTTGGGTCTTCTCAG
ACGTTGGATGGGGTTCTGGCAGATATATCC
ACGTTGGATGGGTTTATTGGAAATGAAGTC
ACGTTGGATGGATCCTACTTACTTCCAGTC
ACGTTGGATGAAGAATGGAAAGTGATGAG
ACGTTGGATGCTAGGCTTGTTCACTATTTG
ACGTTGGATGCACTGAGAGATACAGGAAAG
ACGTTGGATGCTTGTTTCCCCAACATAAGG
ACGTTGGATGGCTTGTTAAATGTGTGTTCC
ACGTTGGATGTCCCTCAGTTTAGTTTTGTC
ACGTTGGATGGATGATGAAAGCATAAGTC
ACGTTGGATGGAGATGTTGCAAAGATGCAAG
ACGTTGGATGAGTGAGACTTAACCGTGGAG
ACGTTGGATGCACCCCCACATTAGCAAAAG
ACGTTGGATGCTTCTTTTCCCTGCATCATC
ACGTTGGATGAGGGAAGTGTTGTAGCATGG
ACGTTGGATGCTGCCTATTCTTCTACGGTC
ACGTTGGATGCAGAAACATGCTTGTAGCAG
ACGTTGGATGCTTTGCTCACAAGAAAGTTGG
ACGTTGGATGCCCCCAAGGCAATGATTTTC
ACGTTGGATGTGGAGGCCACTGGATTAAAG
ACGTTGGATGAGACACAGCTAGCACTTTCC
ACGTTGGATGACTCCCTACCTATCTCTTTG
ACGTTGGATGTCCACAGCCACTGAATAGTC
ACGTTGGATGTGTTACAGCAGCTAGTGTTG
ACGTTGGATGCCTCTAATAGCACCCAGTTC
ACGTTGGATGCACCAGTCCCCTCAAATAAC
ACGTTGGATGGCAGTTCTTAAAGACCTCGG
ACGTTGGATGCCCTAGGATTTTCAGAATGG
ACGTTGGATGGGCTGACTCATTTGTTAGGG
ACGTTGGATGGTGCTTAGGAAATGTTTGTTG
ACGTTGGATGCGTGAATACATGAGAAAGGC
ACGTTGGATGGAGGAGTTATAAGACCTAGAG
ACGTTGGATGACCATATCACAGTTGTTGGG
ACGTTGGATGTATCCATCCTTCAGACACCC
ACGTTGGATGATGGGACAGTAACTGCAGAC
ACGTTGGATGCCTACCTTGCTCTGAGAAAC
ACGTTGGATGCTTCCTGCTTTTAAGCAGTC
ACGTTGGATGTTTTTGGAAATGGCCCAAGG
ACGTTGGATGGAAACAAGTAAATGAGGTCC
ACGTTGGATGAGATCCTCCAGCTCATCTTC
ACGTTGGATGTAATCCTTGGAGGCTCTCTG
ACGTTGGATGAGATTGGTCCCTCACAATGG
ACGTTGGATGATTTGGCCCTGAGGCTTATC
ACGTTGGATGGGAATACATGTGGGTATGTG
ACGTTGGATGATATACGTTGCTTCCTTTGG
ACGTTGGATGAGCCACCAAAACCAAGCTTC
ACGTTGGATGCTTGTAAGGCAGGTCTGATG
ACGTTGGATGTTGTGTGCTATCTTACACTG
ACGTTGGATGACTAGTTGGAATGGGCTTGG
ACGTTGGATGACTGATACCCTACAGTGTGC
ACGTTGGATGGTGCTCAGAGCACTTAAACG
ACGTTGGATGTTGACTCACCCACTTCTGTC
ACGTTGGATGTGTTGATGAGGTGAAGAGGG
ACGTTGGATGTTCAATCAGTCATGCCTGTG
ACGTTGGATGCTAATCTGAAGGCTCCACTG
ACGTTGGATGGCAAGCATCTGCTCTTGAGG
ACGTTGGATGCTGTGTAAAAGAGTTTGAGG
ACGTTGGATGGGTGGAGATGGGATTCTCTG
ACGTTGGATGAACCCAGTCTACACACACAG
ACGTTGGATGGTATCTCCCACTCTTGTACC
ACGTTGGATGCTGGAATACAACATTTCTGG
ACGTTGGATGTTTTTCCTCCTGTACCCTGC
ACGTTGGATGTACATGTGGTTAGAGTCTGG
ACGTTGGATGAAATGCTACTCCAACAGAGG
ACGTTGGATGCTTCATTATCCCCACTGCTG
ACGTTGGATGATCCTGGGCTTTCCTTTGTC
ACGTTGGATGGAGTCTAGTGGACAAGAGAG
ACGTTGGATGAGTGCAACAGAAAAGGCAGG
ACGTTGGATGGGTCCTTGGTATGTGTTCTC
ACGTTGGATGGCATTATGCTAAAGGCTGTC
ACGTTGGATGTCCTCTGATTTAGGCCCTTC
ACGTTGGATGCTATGTTTTCCCCCAGCTTG
ACGTTGGATGGCAAAAGAACAACCACCCAG
ACGTTGGATGGTTTGGTGACTATAGAAACAG
ACGTTGGATGCAGTTTAAAGTCATATTCAC
ACGTTGGATGAACAATAGAGACACACTCCG
ACGTTGGATGTTTAATCCAGGGAGCTCTTC
ACGTTGGATGCACCACTCAGGAAAGCAAAC
ACGTTGGATGAAAATCCCAGTGAAGAGCAG
ACGTTGGATGACAGAAGCACCACAGCTGAG
ACGTTGGATGAGGTTTCCCAAGCTAGACCC
ACGTTGGATGCATCAGCAATATAATGCCGC
ACGTTGGATGTGTGGATCACTGTTCACAGG
ACGTTGGATGGTTCTGGATGTTGGCCATTC
ACGTTGGATGCCACATCATATGCATCTGGG
ACGTTGGATGTCATGTAACCAAGCACCACC
ACGTTGGATGGCTCATTTATAGAAGCAGTC
ACGTTGGATGTTACGACCCAATCACCTTGC
ACGTTGGATGTGTGTCCCCAACCACATTTC
ACGTTGGATGCCTTCTGCTCAACTACCAAG
ACGTTGGATGGCCAAAGACGATGTGGAATG
ACGTTGGATGCCAGTCAAGGAAGCAGTTTC
ACGTTGGATGGTCTGATTAGGCCTAAGAGC
ACGTTGGATGAGGATGCCTGTTGGGTTTTC
ACGTTGGATGATCAGACTTTTCCCAGGCAG
ACGTTGGATGAAGCAGGTACTTACTATGGG
ACGTTGGATGGTACTGTTAGTGTGTCACTC
ACGTTGGATGCTGAGGCACAAGGAGATAAG
ACGTTGGATGACTGACCTGGGTTTGACTTC
ACGTTGGATGTTGCCTAGCCTTACATCCTG
ACGTTGGATGCTCAAAATAGATGATGGACTG
ACGTTGGATGGACCTTCCTGTTCCTAGATG
ACGTTGGATGTGACTGGACTGTGACATAGC
ACGTTGGATGAATCTTGGAGCCTTGGAGAC
ACGTTGGATGGTGCTTCTCACAAAAGCCTG
ACGTTGGATGATCCATCTCTGTCAGAGTTC
ACGTTGGATGAGAGAACTGACCCTTCACTG
ACGTTGGATGGAGAGAGGGAGAAAGTAGAG
ACGTTGGATGCCCTTACTCAGTGATTCCTC
ACGTTGGATGTTTAATAGGGAAAGTATTGG
ACGTTGGATGCACACCCAGAAGCACTGATA
ACGTTGGATGTACCATGCTCATTGAACTCG
ACGTTGGATGGGAGATTTGATAGGAAGTGC
ACGTTGGATGGCCCATCCTTCACTAACTTG
ACGTTGGATGAACAGTGGTGGCCCATCAGT
ACGTTGGATGCTGCCAGGGAATAGGAGATG
ACGTTGGATGCGCTGAAAGAGACACTGAAG
ACGTTGGATGTTACAGTTGAGAGCCACTGC
ACGTTGGATGTGCCATTCATTGCTCTACAC
ACGTTGGATGAAGTCATTTGAGGCCCATCC
ACGTTGGATGCACTTCCAGCTGCTGCTTTC
ACGTTGGATGTCTAGGTTGAGACTCAGGTG
ACGTTGGATGGGTTTAAGCAACATGAAAGC
ACGTTGGATGTACGACCAGAATGGAAGGAG
ACGTTGGATGATTGAACCCTGGGAAGGTGG
ACGTTGGATGACTTAACCCCCAGTGTGATG
ACGTTGGATGCACTTATCCCATTCACGAGG
ACGTTGGATGACTCTCACACAAAGTTTGCC
ACGTTGGATGCGGTATTGTCTTAAGACTGA
ACGTTGGATGGTCAGAGATTTCTGCCTAAG
ACGTTGGATGCATCTGCCATGATGATCCTG
ACGTTGGATGACCCAGGTGACCGAATAAAG
ACGTTGGATGGTAGCTACGTTCTTTGGAGG
ACGTTGGATGGAGCTTCTATGAAACGTGTG
ACGTTGGATGGGTTTCTGCCAAAAACCTTG
ACGTTGGATGCAGTCTATCTCTTGCTCTAC
ACGTTGGATGGAAGACCATTATGTTTCTGAC
ACGTTGGATGCAGCAGCCTTTGAAAGACAC
ACGTTGGATGTCAGCAGCTTACGGTTTCAG
ACGTTGGATGCTGGTTCTGTGAAATAAGAC
ACGTTGGATGTGGTGTCTTTACCTCTTTAC
ACGTTGGATGGGTCACTAGTGTATATTTTG
ACGTTGGATGGACTCCCAACACACAATACC
ACGTTGGATGCAAAAGAAGCTGGATTGCTC
ACGTTGGATGCCAAGAAAGGCAATGTTGGG
ACGTTGGATGGAGGAGAAGGTGATGTGAAG
ACGTTGGATGCCCTGGAGTTCCTTTTCTTG
ACGTTGGATGATCCCTCATTCTTTCTCCAC
ACGTTGGATGGAGAAGCTGAGGAAGCAAAG
ACGTTGGATGATGCTAAGGATTCTGGGGTC
ACGTTGGATGCATATGCTAAGAGCCAGGAC
ACGTTGGATGGGGCATGACACAACTCAAAC
ACGTTGGATGACCTGAGTTTTCAGCCGTTG
ACGTTGGATGAACAGAGGGTTTAACAGCAC
ACGTTGGATGACCTTACTCAGTTCTATTC
ACGTTGGATGCCAATATTTTTTCCCTAGGT
ACGTTGGATGTGTGCATTAAATCCTCCCCC
ACGTTGGATGTAAGCTCCCCCATCCAAGAC
ACGTTGGATGCACAGGTCTACCTTGATTTC
ACGTTGGATGTATCTGGGTCATTGTAAGGC
ACGTTGGATGTTCCCAAACATAATCACAG
ACGTTGGATGAGGGCACAAAGACATCAAAG
ACGTTGGATGGGCATAGCGCCTGTGCTTAA
ACGTTGGATGGGCAGGCACTCTATCAATAC
ACGTTGGATGTGTTTGTTGCTGCGTGCTTC
ACGTTGGATGAGGGTATAGGAAACAGCTTC
ACGTTGGATGATCCTCTCTCCTAACACCAG
ACGTTGGATGCATTTACCCACAAAGGTAAG
ACGTTGGATGTAGTCCCTGACATTGGAGAG
ACGTTGGATGGATGCAGAATAAGCATTTGAC
ACGTTGGATGGAGGCACTTTTTTCTGTTCC
ACGTTGGATGCTGTCTCAAGTGTCTGGTTC
ACGTTGGATGATCCATCCACCCATCCATTG
ACGTTGGATGGTCCTAAGTTAAAAGAATGG
ACGTTGGATGCTCATGCCGACAAAACTTCC
ACGTTGGATGATCAAGAGGAAAATGGACAG
ACGTTGGATGGATTTACTCAACTCTCTGGG
ACGTTGGATGTCTCCCCACTTTGTTCTGAG
ACGTTGGATGTCAACTAAAGGGCAGTAACC
ACGTTGGATGACTGTGCCTGGACAAAGAAG
ACGTTGGATGTAGCACCAGGCTTACTAGAC
ACGTTGGATGGACAGTTTTTAAATCTTTTAC
ACGTTGGATGCTGTATTCCCATACTACTTG
ACGTTGGATGTGAGTGATAGGTCCTCTCTG
ACGTTGGATGAGCTCACAAAACTAACACAC
ACGTTGGATGCAAAAGAACCTGGCTCATGG
ACGTTGGATGATATGTCACGCATAGCCCAG
Conditions Used for Testing
PCR
PCR was performed with or without the addition of Tsp509I to the PCR cocktail mix as indicated in Table 12. PCR cycling was performed for all samples with the cycling conditions in Table 13 to allow Tsp509I digestion of the DNA immediately prior to PCR amplification and in a single tube. This was used even if there was no Tsp509I added to the cocktail.
SAP
SAP dephosphorylation was carried out with standard conditions including the SAP cocktail preparation below in Table 14.
TypePLEX Extend
TypePLEX Extend reaction was carried out with standard conditions including extend cocktail preparation below in Table 15.
Digestion of Heterozygous SNPs in Genomic DNA
CEPH genomic DNA obtained from the Coriell collection was used to test the ability of Tsp509I to specifically digest one allele of each SNP. The informative allele peak area ratios of DNAs heterozygous for the indicated SNPs were determined. The informative allele, alternatively called the target allele, is defined as the allele NOT recognized by Tsp509I enzyme. Tsp509I treatment significantly increased the peak area ratio. With no Tsp509I treatment, heterozygous DNAs show median allele ratios ranging from 0.4-0.6 depending on the SNP. After Tsp509I treatment, for the majority of heterozygous DNAs, the median peak area ratio is above 0.8 with many peak area ratios at 1.0. Peak area ratios of 1.0 indicate that there is no detectable non-informative (i.e., non-target) allele peak area present.
2% Mixture Model
A DNA mixture model was prepared from CEPH genomic DNA obtained from the Coriell collection. The DNA mixture model was used to test the ability of Tsp509I to enhance the detection of one allele of a SNP when present at a low fractional concentration. Briefly, the DNA mixture model comprises 47 unique child/maternal DNA pairs mixed together such that the child's DNA (the low fractional concentration DNA) is present at only 2% of the total DNA. For the studies here, DNA was added to the PCR such that there were 20 genomic copies of the low fractional concentration DNA and 980 copies of the high fractional concentration DNA in each PCR. In these mixture studies, not all DNA pairs will yield informative data for every SNP. Informative data can only be obtained for a SNP when the maternal genotype is homozygous for the non-informative allele and the child's genotype is heterozygous for the SNP. With no Tsp509I treatment, potentially informative DNA mixtures show median informative peak area ratios at background levels. After Tsp509I treatment, the majority of DNA mixtures with potentially informative genotype combinations for the indicated SNP show median peak area ratios above 0.5 with many peak area ratios at 1.0. Peak area ratios of 1.0 indicate that there is no detectable non-informative allele peak area present. This indicates the utility of the multiplexed SNPs to detect a low fractional concentration DNA present at least as low as 2% of the total DNA present and at levels as low as 20 genomic copies of DNA.
Detection of Low Fractional Concentration DNA
Modified versions of multiplexes 2, 5, and 6 with a total of 95 SNP assays (see Table 16) were tested for their ability to detect a low fractional concentration DNA. Sample test groups included:
For the comparison, each of the above sample types was digested with Tsp509I prior to genotyping with the TypePLEX extend assay. Separately, maternal genotypes from undigested maternal DNA was determined to identify potentially informative SNPs for each sample. For this analysis, no genotype information obtained directly from child genomic DNA or fetal genomic DNA was used.
With 95 SNP genotype assays, one would expect to have 3 or more informative genotype combinations in ˜99.9% of cases with biologically related maternal and child genotypes. Therefore, detection of at least 3 informative SNP alleles present in a Tsp509I digested sample that are not present in an undigested maternal only DNA sample should allow detection of a low fractional concentration DNA. Increasing this required number of detected informative SNP alleles to greater than 3 will likely increase the specificity but at the expense of sensitivity.
In prior studies with the DNA mixtures, it was noted that in the Tsp509I digested samples, background levels of informative allele peak area could lead to artificially high detection of an informative allele peak area ratio. Therefore, preliminary threshold criteria were established to improve the accuracy of detecting informative SNP alleles arising from low fractional concentration DNA. In the data here, these thresholds are defined as follows:
The criteria used here to determine the presence or absence of an informative SNP allele are preliminary and are only exemplary. Additionally, individual SNP assays within the multiplexes may have their own criteria. Alteration of these criteria can have significant impact on the detection of informative SNP alleles in either a positive or negative manner.
As can be seen in
Detection of Fetal Identifier Alleles in Maternal Plasma
The ability to detect fetal identifier alleles in maternal plasma DNA and non-pregnant female plasma DNA was compared. Ninety-two of the fetal identifier SNPs in Table 16 in 3 multiplexes were assayed by genotyping buffy coat, PBMC or whole blood genomic DNA from plasma samples. The samples were analyzed in parallel with and without Tsp509I digestion, and they were subsequently genotyped for the same SNPs. Genotype measurement was performed on the MassARRAY® system. A fetal identifier allele was counted as ‘detected’ if the undigested genomic DNA was homozygous for the cleavable SNP allele and the matching plasma DNA sample showed the presence of the non-cleavable SNP allele after digestion of the plasma DNA with Tsp509I.
As expected, the fetal identifier alleles were detected in the pregnant maternal samples and not the non-pregnant plasma samples. As the number of SNPs tested increases, the probability of the number of informative SNPs also increases. This is shown graphically in
The entirety of each patent, patent application, publication and document referenced herein hereby is incorporated by reference. Citation of the above patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents.
Modifications may be made to the foregoing without departing from the basic aspects of the invention. Although the invention has been described in substantial detail with reference to one or more specific embodiments, those of ordinary skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, yet these modifications and improvements are within the scope and spirit of the invention.
The invention illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising,” “consisting essentially of,” and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and use of such terms and expressions do not exclude any equivalents of the features shown and described or portions thereof, and various modifications are possible within the scope of the invention claimed. The term “a” or “an” can refer to one of or a plurality of the elements it modifies (e.g., “a reagent” can mean one or more reagents) unless it is contextually clear either one of the elements or more than one of the elements is described. The term “about” as used herein refers to a value within 10% of the underlying parameter (i.e., plus or minus 10%), and use of the term “about” at the beginning of a string of values modifies each of the values (i.e., “about 1, 2 and 3” is about 1, about 2 and about 3). For example, a weight of “about 100 grams” can include weights between 90 grams and 110 grams. Further, when a listing of values is described herein (e.g., about 50%, 60%, 70%, 80%, 85% or 86%) the listing includes all intermediate values thereof (e.g., 54%, 85.4%). Thus, it should be understood that although the present invention has been specifically disclosed by representative embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and such modifications and variations are considered within the scope of this invention.
Certain embodiments of the invention are set forth in the claims that follow.
This patent application is a continuation of U.S. patent application Ser. No. 12/411,329, filed on Mar. 25, 2009, which claims the benefit of U.S. Provisional Patent Application No. 61/039,747, filed on Mar. 26, 2008, entitled RESTRICTION ENDONUCLEASE ENHANCED POLYMORPHIC SEQUENCE DETECTION. This patent application also is related to U.S. Provisional Patent Application No. 60/908,167, filed on Mar. 26, 2007, and Patent Cooperation Treaty International Patent Application No. PCT/US2008/058317, filed on Mar. 26, 2008, and published as Publication No. WO2008/118988 on Oct. 2, 2008, each entitled RESTRICTION ENDONUCLEASE ENHANCED POLYMORPHIC SEQUENCE DETECTION. The entirety of each of these three patent applications is hereby incorporated herein by reference.
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