Patent Application
20100086918
The invention relates to assay methods for identification of polymorphisms in genes, especially deletions or translocations in genomic DNA. The invention further relates to identification of chromosomal anomalies arising from such polymorphisms in mammalian cells.
Sequence variations in genomic DNA from wild-type, such as gene deletions, are often associated with the onset and progression of primary cancers. For example, deletions of the CDKN2A gene coding for the p16INK4a and p14ARF proteins commonly occur in human cancer cell lines. However, the size of the deletions, and therefore the location of their breakpoints, vary widely.
The diagnostic and cancer monitoring potential of deletions from genomic DNA has been difficult to exploit clinically because (a) tumor specimens are invariably contaminated with normal cells, demanding time consuming methods for tumor nucleic acid extraction, and (b) the sizes of deletions in particular can vary from <1 to >40 kb. Currently available methods for deletion mapping (including Southern blotting, LOH analysis, fluorescence in situ hybridization, real time PCR and array based comparative genome hybridization [CGH]) all suffer from various technical limitations and, consequently are not able to detect many deletions, nor to precisely characterize them.
If a protein product of a gene is ubiquitously expressed, immunohistochemical (IHC) detection of the protein can be used as a screening surrogate for genetic or epigenetic gene inactivation. However, the production of many cancer-related proteins, such as p15INK4b, p16INK4a, and p14ARF, varies with cell differentiation, growth and senescence. Further, where the cancer-related sequence (in genomic DNA or a fusion transcript) is not known in advance, existing detection protocols require the gene sample to be substantially (e.g., 80%) pure. Accordingly, IHC and other existing techniques for analysis of these proteins has not been an accurate screen for assessing deletions or sequence variations in the coding gene (e.g., for p16/p14 and p15).
A need, therefore, exists for a method that will enable detection of even small gene deletions in the presence of a vast excess of wild-type gene (e.g., from non-isolated primary tumors). With such a method in hand, breakpoint-specific molecular probes for use in personalized monitoring of cancer progression in individuals may be developed.
The invention provides a multiplex PCR-based method for detecting a polynucleotide having a nucleotide sequence differing from the wild-type polynucleotide sequence of a gene, wherein the variation is a deletion, translocation, inversion or fusion of nucleotides, and the variant polynucleotide is in the presence of an excess of the wild-type molecule. More specifically, the detection is of a polymorphism in genomic DNA, and is accomplished directly and/or may be confirmed by detection of a corresponding abnormal RNA fusion transcript.
The multiplex PCR method is particularly advantageous in that it allows for identification and characterization of deletion segments and their breakpoint boundaries, even against a background containing a vast excess of the wild-type molecule; e.g., at least a predominance (>50%) of wild-type and beyond the limits of conventional assays, such as IHC (e.g., ≧80%). For example, the method allows for detection of chromosomal gene deletions and mapping their breakpoints in samples of genomic DNA containing up to about 99.9% wild-type DNA contamination. In another embodiment, the presence of an abnormal RNA fusion transcript in a sample was detected in a sample containing ˜3000 times wild-type RNA.
To this end, multiple primer pairs approximating the flanking sequence of a deletion sequence are subjected to multiplex PCR. Each number of the primer pair is spaced about ≧1 kb from the other member, or may be placed closer together in embodiments of the invention utilizing a poison primer. Forward and reverse primer pairs are provided and separated into groups (up to about 100 primers per group) for use in multiplicity of multiplex PCR reactions, comprising a primary amplification step. A secondary amplification step may be performed to increase the product specificity for the boundaries of the sequence variation. To target relatively small (<1 kb) deletions, poison primer PCR using a primer pair external to the variant segment and a third primer internal to the segment will be utilized to target the deleted sequence.
The invention further provides means for determining the susceptibility of an individual organism, such as a mammal (and particularly a human), to develop a disease clinically related to the occurrence of deletions, inversions or translocations in genomic DNA, such as cancer, as well as diagnosing and monitoring the progression of such a disease in an individual by tracking and comparing the occurrence of targeted deletions or translocations in different populations of cells, or in the same population of cells over time. For example, with knowledge of genomic breakpoints or the identity of related fusion transcripts, molecular probes are developed to inform the clinician of the presence of a sequence variation in genomic DNA or RNA transcripts with pathological implications for the onset and progression of cancer.
To this end, a first embodiment of the invention provides multiple primer pairs that are hybridizable to a target polynucleotide (e.g., one or more chromosomal gene segments), where each number of the primer pair is spaced≧about 1 kb from the other member. Forward and reverse primer pairs are provided and separated into groups (up to about 100 primers per group) for use in multiplicity of multiplex PCR reactions, comprising a primary amplification step. Advantageously, the array reactions are suitable to automation by separation of the primer pair groups into wells of a microtiter plate.
Of the multiplex PCR products, few (generally, one or two) will span the deletion, translocation or inversion boundary, since the other primer pairs should be spaced too far from the boundary for efficient amplification.
Preferably, the primary boundary-spanning amplification products (amplicons) will be further amplified to increase the product specificity for the target boundaries. Nested PCR methods are particularly useful for use in this secondary amplification step. To target relatively small (<1 kb) deletions, poison primer PCR using a primer pair external to the targeted deletion segment and a third primer internal to the segment will be utilized to target the deletion in the wild-type genome.
To characterize the boundaries of a genomic deletion, translocation or inversion, or to identify an abnormal fusion transcript, sequence analysis is performed on the amplicons obtained from the primary or, if performed, secondary amplification steps. In one embodiment of the invention, the analysis step is performed on a genomic tiling array. According to this embodiment, amplicons indicative of the boundaries of a sequence variation, such as deletion breakpoints, obtained from the PCR step(s) of the invention are labeled for hybridization on one or more gene-specific tiling arrays. The boundaries of the targeted sequence variation are considered confirmed by probe hybridization to the putative breakpoints identified in the PCR step(s).
In an alternative embodiment of the invention, the sequence variation is characterized by direct sequencing according to conventional techniques.
In a further alternative embodiment of the invention, the primer pair groups prepared for multiplex PCR are not separated physically into wells before amplification. Instead, the primer pairs and template polynucleotides are separated into groups by admixture in a water-in-oil emulsion. Most preferably, the primers are bound to a solid phase support, such as nanoparticles prior to admixture into the water-in-oil emulsion. Amplification may therefore be performed in a single tube rather than a multiwell plate.
In an additional variation on the invention, probes specific to one or more sequence variations detected according to the assays of the invention are developed. Such probes allow for determining the susceptibility of an individual to develop a disease clinically related to the occurrence of genomic deletions, translocations or inversion, such as occur in certain cancers or heart disease conditions. The methods of the invention also permit diagnosis and monitoring of the progression of such a disease; e.g., as measured by changes in the length, size or number of polymorphisms in the target nucleic acid, especially genomic DNA.
In its broadest sense, the present invention allows the detection and characterization of any polymorphism in, or deletion of, a target nucleic acid sequence of diagnostic or therapeutic relevance, where the target nucleic acid sequence is present in a biological cell sample from any organism, such as the margins of a primary tumor or a regional lymph node. Thus, the target nucleotide sequence may be, for example, deletions, translocations or inversions in genomic DNA, or exon junctions in corresponding fusion RNA transcripts. Further, the method of the invention can be used to detect and characterize multiple target polynucleotides; e.g., multiple deletion segments.
With respect to deletion segments in particular, the invention exploits the fact that a nucleic acid sequence from which a polynucleotide segment has been deleted is shorter than, and therefore should be preferentially amplified compared to, the longer wild-type sequence using “approximated” flanking primers (
I. Overview of PAMP Methodology.
The invention adapts and utilizes techniques for PCR amplification of DNA in a biological sample. The basic techniques for performing PCR are well-known in the art. For further details, consult U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis, et al., the disclosures of which are incorporated herein.
Primer approximation PCR techniques have been previously used to isolate deletion variants in C. elegans (Jansen, et al, Nat. Genetics, 17:119-121, 1997). The method relies on identifying a single band that is the product of a successful PCR when a pair of specific primers is brought together by deletion, on an agarose gel. However, the procedure can yield ambiguous results since only deletions near single primer pairs can be identified.
Multiplex PCR enables generation of multiple amplicons in a single PCR reaction, and is especially useful in amplifying nucleic acids of known sequences (see, e.g., Boriskin, et al., J. Clin. Microbiol., 42: 5811-5818, 2004). The existing method is not especially useful, however, for detecting deletion segments of unknown sequence or length.
The Jansen, et al. and Boriskin, et al. papers are incorporated herein by this reference to illustrate the general application of the primer approximation and multiplex PCR techniques in the art before the invention.
The PAMP approach of the invention combines, adapts and refines the general principles of primer approximation PCR and multiplex PCR, as illustrated in
A representative protocol for each primer approximation reaction (multiplied for multiplex PCR) is described in Example II. In general, only one or two pairs of PCR reactions will produce specific PCR products spanning the deletion boundary, since the other primer pairs should be too far from the breakpoint for efficient amplification.
The multiplex PCR conditions may be varied by those of ordinary skill in the art (e.g., according to the number of primers used, the extent of automation, the polymerases applied, and the like), but are essentially as described in Boriskin, et al., J. Clin. Microbiol., 42:5811-5818, 2004, and in Example I, using 96-well plates. Each plate will preferably be used for 1 cell line or tumor sample. Further, although there are multiple possible combinations of the primer groups (100 being the example shown in
The primary amplification step itself can be rendered semi-automatic by using commercially available robots for liquid handling, addition of reagents and the like, such as the BIOMEX FX™ from Beckman-Coulter. A particular application of the primary multiplex PCR step of the invention is illustrated in Example II.
Secondary groups of different primers can be used to increase the specificity and further amplify the products from the first multiplex PCR reactions (equivalent to internal primers for nested PCR). Then aliquots from each PAMP can be mixed to hybridize on a single genomic tiling array. Unlike traditional array CGH, only spots representing genomic sequences near the breakpoints will light up on the array (see, schematic in
If a secondary amplification is performed to increase the specificity of the amplicons for deletion breakpoints, nested PCR utilizing additional primers hybridizable to the boundaries of the deleted gene segments will be performed (see, e.g., Example II). General techniques for performing nested PCR are well known in the art.
Briefly, nested PCR uses two sets of amplification primers. The target nucleic acid sequence of one set of primers (termed “inner” primers) is located within the target sequence of the second set of primers (termed “outer” primers). In practice, a standard PCR reaction is first run with the patient sample using the “outer primers”. Then a second PCR reaction is run with the “inner primers” using the product of the first reaction as the amplification target. This procedure increases the sensitivity of the assay by reamplifying the product of the first reaction in a second reaction. The specificity of the assay is increased because the inner primers amplify only if the first PCR reaction yielded a specific product.
II. Poison Primer Adaption of PAMP Methodology.
While the PAMP approach works well for larger genomic deletions, it is less discriminating for smaller deletions (i.e., less than approximately 1 kb). However, by employing a “poison primer” nested PCR strategy for the secondary amplification step, it is possible to insure that even very small deletions are selectively amplified (see, e.g. Edgley, et al., Nucleic Acids Res., 30:e52, 2002, incorporated herein by this reference;
The amplicons from smaller deletions are not sufficiently different from wild-type to provide a competitive advantage in PCR. However, when a “poison primer” from the deleted sequence anneals to the wild-type genome, it competes for the amplification of the WT genome with the common set of primers, which amplifies both wild-type and variant genomes. The amplification reactions with poison primers in wild-type DNA are favored, since the PCR products are smaller.
Briefly, a third functional PCR primer that falls between the two external primers flanking a deletion segment identified in the primary amplification step is designed (
In a further round of PCR, two primers are placed just inside the external first round primers. The shorter wild-type band from the first round cannot serve as a template for the second round PCR because it does not include one of the second round primer sites. The longer wild-type fragment can serve as a template, but because its production is limited by competition in the first round, its production in the second round is limited correspondingly. The lower level of effective wild type gives the deletion fragment an advantage since the majority of the primary PCR products are poison primer derived, and lack one of the secondary primer annealing sequences (Example V). The extension of this methodology to a multiplex format yields the PPMP (poison primer [approximation] multiplex PCR) method of the invention.
III. Primer Autopairing Via Emulsification (PAVE).
An alternative amplification method in which use of microtiter plates is not required involves compartmentalization of primer pairs, templates and a solid phase support (e.g., microparticles or beads). The approach, known in the art as water-in-oil emulsion PCR is generally described in Diehl, et al., Natl. Methods, 3:551-559, 2006; Kojima, et al., Nucleic Acids Research, 33(17):e150, 2005; and Shendurne, et al., Science, 309:1728-1732, 2005 (all incorporated herein by this reference), and is illustrated by the schematics in
Briefly, the primers are carried by nanoparticles that are diluted to two nanoparticles per compartment on average through water-in-oil emulsification, to produce droplets containing other PCR reagents and the template nucleic (
The primers containing nanoparticles can be manufactured with various techniques, for example by another warm oil-in-water technique as described in Oyewumi et al. Drug Development and Industrial Pharmacy 28:317-328, 2002. Briefly, each group of primers in solution is mixed into melted emulsifying wax in the presence of an emulsifier, at 55° C. Wax nanoparticles, which average 100 nm in diameter, form when the reaction is cooled to room temperature.
IV. Microarray Analysis of Polymorphisms Identified According to the Assays of the Invention.
For sequence analysis of polynucleotides of interest, genomic tiling microarrays of varying formats have been developed in the art (see, e.g., Liu, Y. T., et al., Clinical Infectious Diseases, San Diego, p. 196, LB-3, “A virus-specific DNA microarray as a diagnostic and discovery tool,” 41st Ann. Meeting of ISDA, 2003; Wang, et al., PNAS USA, 99:15687-15692; 2002; Wang et al. PLoS Biol., 1:E2, 2003; Ishkanian, et al., Natl. Genet., 36:299-303, 2004). Compared to the present cost of direct sequencing, microarray characterization of polynucleotides is relatively cost-effective, and can be readily automated. The use of such arrays is generally illustrated by the schematics in
For analysis of boundaries about a deletion, translocation or inversion of one or more nucleotides in a gene, a locus array for the target gene is prepared. For example, a tiling array with an average probe length of 1 kb will cover a 500 kb region. Shorter probes (50-70 bp) should be utilized for analysis of gene sequences with numerous repetitive motifs, such as are found in the CDNK2A/B loci (see, e.g., Bertone, et al. Genome Res., 16:271-281, 2006, incorporated herein by this reference). An assay according to the invention is performed on the array and scanned; e.g., using a commercially available scanner such as the GENEPIX™ 4000B from Axon (see, e.g., Eisen, et al., Methods Enzymol., 303:179-205, 1999). Targeted boundaries are identified in scans as spots with high signals, as illustrated in, for example,
V. Diagnostic and Therapeutic Monitoring Using Breakpoint-Specific Probes.
Once a deletion segment or corresponding fusion transcript has been characterized, probes can be developed to target them in cells obtained from the same patient. This allows clinicians to practice personalized medicine; e.g. cancer therapy, by monitoring the progression of the patient's cancer (such as by recognizing when the size of a deleted segment is altered or when multiple deletions or translocations occur) or treatment (e.g., if the affected chromosomal region is stabilized).
With knowledge of the boundaries of the sequence variation in hand, the information can be used to diagnose a pre-cancerous condition or existing cancer condition. Further, by quantitating the number of cells in successive cell samples which bear and acquire the deletion or other polymorphism at separate locations in the body and/or over time, the progression of a cancer condition can be monitored. For example, data provided by assaying the patient's tissues at one point in time to detect a first set of sequence variations from wild-type could be compared against data provided from a subsequent assay, to determine if changes in the location, size or number of sequence variations have occurred.
A highly specific adaptation of nested PCR that is particularly preferred technique for quantitating cancer burden with identified signature breakpoint sequences as described in U.S. Pat. No. 5,747,251, the disclosure of which is incorporated herein by this reference and detailed in Example III. Briefly, the technique of the '251 Patent involves competitive PCR is performed using a competitor template containing an induced sequence variation of one or more base pairs which results in the competitor differing in sequence (but not size) from the target template. One of the primers is biotinylated or, preferably, aminated so that one strand (usually the antisense strand) of the resulting PCR product can be immobilized via an amino-carboxyl, amino-amino, biotin-streptavidin or other suitably tight bond to a solid phase support which has been tightly bound to an appropriate reactant.
The bonds between the PCR product, solid phase support and reactant will be covalent ones, thus reliably rendering the bonds resistant to uncoupling under denaturing conditions. Once the aminated or biotinylated strands of the PCR products are immobilized, the unbound complementary strands are separated in an alkaline denaturing wash and removed from the reaction environment. Primers corresponding to the target and competitor nucleic acids are labeled with a detection tag. The primers are then hybridized to the antisense strands in absence of competition from the removed unbound sense strands. Appropriate assay reagents are added and the degree of hybridization is measured by ELISA measurement means appropriate to the detection tag and solid phase support means used, preferably an ELISA microplate reader. The measured values are compared to derive target nucleic acid content, using a standard curve separately derived from PCR reactions amplifying templates including target and competitor templates.
Where a deletion or other polymorphism is found in an individual mammal who has not yet developed symptoms of a disease clinically related to the presence of such deletion or polymorphism, such as cancer, the deletion or polymorphism will be indicative of a genetic susceptibility to develop the cancer condition. Analysis data obtained by performance of the methods of the invention will be of particular prognostic value where the abnormality is carried in germline cells and/or has the individual has a family history of a particular cancer condition.
Where other indicia of the presence of the disease in the individual are present, such as clinical symptoms, biopsy results, positive radiological examinations or the like, analysis data obtained by performance of the methods of the invention indicating the presence of a deletion or translocation of one or more nucleotides in genomic DNA clinically related to the occurrence of the disease will also be of particular diagnostic value.
A determination of susceptibility to disease or diagnosis of its presence can further be evaluated on a qualitative basis based on information concerning the prevalence, if any, of the cancer condition in the patient's family history and the presence of other risk factors, such as exposure to environmental factors and whether the patient's cells also carry a deletion of another gene; e.g., for both CDKN2A and MTAP, as occurs in many primary cancers. Multiple gene deletions and translocations of the kind that occur in connection with the CDKN2A and MTAP coding sequences are of particular diagnostic and cancer monitoring utility.
For example, as described in U.S. Pat. No. 6,689,561 (the disclosure of which is incorporated herein by this reference), screening of cells from a known leukemia cell line (U937; ATCC Accession No. 1593) indicates that they contain an intragenic microdeletion of 18 base pairs in the CDK4I5′ exon (see, '561 Patent at Example VI). Using such information and the techniques for identifying sequence variations in genes which are illustrated herein, those of ordinary skill in the art will be able to screen cell samples from particular 9p21-linked tumors for reproducible polymorphisms and/or deletions of CDK4I to determine genetic susceptibility to, as well as the existence of a cancer condition as defined herein (particularly melanomas, gliomas, non-small cell lung cancers and leukemias).
TMPRSS2:ETS gene fusions are also a recurrent prostate cancer-specific event. Among all of the reported fusion partners in the ETS family of genes, TMPRSS2:ERG is the most prevalent one, variants of which have been associated with progressive diseases. As described in Examples VII and VIII, TMPRSS2:ERG fusion transcripts were detected with total RNA from 3 cells containing the fusion in the presence of more than 3000 times excess of background RNA and in a primary prostate tumor having no more than 1% of cancer cells. The ability to detect multiple transcript variants is critically dependent on both the primer and probe designs. The methods of the invention will therefore facilitate clinical studies of transcript RNA and can be readily adapted to include other fusion genes.
The invention having been fully described, aspects of its practice are illustrated by the examples below. The scope of the invention shall not, however, be limited by the examples, but is instead defined by the appended claims on issuance of this application, or any applications which claim the priority of this application. Standard abbreviations are used in the examples, such as “ml” for milliliters, “min” for minutes, and the like.
The invention may be applied to any gene or other polynucleotide in which at least a portion of the primary sequence outlying a potential deletion, translocation or inversion region is known, so appropriate primer pairs can be developed to hydridize to the target molecule at loci a distance apart; e.g., ≧1 kb apart for application of the PAMP method, or at loci <1 kb apart for the PAMP/poison primer embodiment of the invention. For purposes of illustrating application of the invention to genomic DNA, the examples herein are of use of the invention to characterize deletions in the CDKN2 region on human chromosome 9p21. The CDKN2 region experiences homozygous deletions in a diverse range of cancer cell lines, and so is a exemplary target molecule to demonstrate the use and sensitivity of the invention. However, those of ordinary skill in the art will understand that application of the invention is not limited to the CDKN2 region of human chromosome 9p21, or to any particular chromosome or polynucleotide, or to genomic DNA of any particular species.
The CDKN2 region on human chromosome 9p21 encodes three different tumor suppressor genes (
Homozygous deletions of chromosome 9p encompassing the CDKN2A region are very common in cancer cell lines of diverse origin, including lines derived from tumors of the lung, bladder, brain, head and neck, ovary, pancreas, skin, and blood, a finding later confirmed in many types of primary tumors. In addition, CDKN2A inactivation reportedly happens early during cancer development in some well documented solid tumors, including pancreatic adenocarcinoma, head and neck squamous cell cancer and esophageal cancer.
To cover a 500 kb genomic sequence on chromosome 9p21 flanking the CDKN2A locus, 500 primary primers (250 pairs) were synthesized and divided into 20 groups, each with about 25 primers. 10 primer groups were forward (F1-F10) and 10 were reverse (R1-R10), as shown in
For secondary amplification (to increase the specificity of the PCR reactions and further amplify the PCR products from multiplex PCR reactions performed with the primary primers), secondary groups of primers were developed.
Primer sets can be selected with Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) or GeneRunner (http://www.generunner.com/) avoiding repetitive sequences, which are predicted by Repeatmasker (http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker). The specificities of the PCR primer pairs may also be evaluated by in silico PCR (http://www.genome.ucsc.edu/cgi-bin/hgPcr?command+start). Alternatively, primer design may be assisted by the optimization technique described in co-pending, commonly assigned U.S. Provisional Patent Application No. 60/931,793, filed Mar. 25, 2007.
A simplified PAMP scheme is shown in
Groups of forward and reverse primers (generated as described in Example I) were used to generate amplicons from 100 ng of genomic DNA templates for multiplex PCR (conditions: 35 cycles of 92° C., 30 seconds; 55° C., 2.5 minutes). The products were subsequently used as templates for another round of amplification with the same PCR protocol except replacing dTTP by a 4:1 mixture of aminoallyl dUTP (Ambion, Austin, Tex.) and dTTP for probe labeling.
For ease of analysis, an INK4A exon 1-2 minigenomic tiling array was created to cover a 25 kb fragment in the CDKN2A locus (see,
For the array, DNA probes were generated by PCR on non-repetitive genomic sequences with BAC clone RP11-14912 (obtained from BACPAC Resources Center at Children's Hospital Oakland Research Institute, Oakland, Calif.). The template probes were printed on poly-L-lysine slides at 0.1 mg/ml. Human Cot-1 DNA (Invitrogen, Carlsbad, Calif.), which is enriched for repetitive sequences, and herring sperm DNA (Promega, Madison, Wis.), which was used as nonspecific control, were also spotted on the array as described by the manual of the commercially available DeRisi™ arrayer.
Only spots with probes close to the breakpoints hybridized to the amplicons when Detroit 562 genomic DNA was used as a template (
In addition, four separate arrays were used to hybridize the individual PAMP products described above. Only F2-R1 produced the same result as shown in
Secondary PCR was performed using the nested PCR with pairs of specific primers designed according to the earlier PAMP results. The PCR product was labeled for array hybridization, yielding a result very similar to that shown in
To mimic the heterogenous population of cancer and host cells typically found in solid tumors, various genomic DNA ratios of Detroit 562 (variant) and HEK293 (wild type) were used as templates for PAMP and array hybridization. CDKN2A deletion was detected when only 2% of variant genomic DNA was present in these experiments. The array result with contaminated DNA was as clean as in
The same approach was used to map the breakpoint of a breast cancer cell line (Hs 578T), quickly yielding a result consistent with another report (Sasaki, et al., Oncogene, 22:3792-3798, 2003).
The solid phase capture PCR methodologies described in U.S. Pat. No. 5,747,251 are particularly useful alternatives to standard nested PCR techniques as described in Example II. The disclosure of the patent is incorporated by reference herein; briefly, the methods described are summarized as follows:
A. Performance of Method.
Conventional or anchored PCR is performed to coamplify the target and competitor templates using the modified and unmodified primers. The PCR products may be purified by minicolumn (using, for example, the MAGIC PCR PREPS™ product from Promega, Madison, Wis.). The resulting products will consist of antisense strands having the coupling agent attached thereto and sense strands without coupling agent.
Immobilization or capture of antisense strands is performed by placing a diluted aliquot of the double-stranded PCR products onto the solid phase support (e.g., coated ELISA plate wells). The PCR products are allowed to stand in the plate wells in the presence of a coupling reagent for a period of time sufficient for capture of the antisense strands bearing the coupling agent to the reagent coating each well. Sense strands are then separated from the captured antisense strands and removed from the solution in each plate well by incubation with an alkaline denaturing salt (such as 0.1N NaOH) and washing with a buffer solution.
After removal of the unbound sense strands, the labeled probes are added to each plate well and hybridization is allowed to occur with the captured antisense strands. A substrate, antibody or other assay reagent appropriate to interact with the label used on the probes is added to each plate well and the reaction stopped at an appropriate point. An ELISA microplate reader (such as the THERMO MAX™ microplate reader from Molecular Devices of Menlo Park, Calif.) is used to measure absorbance in each well and the values compared to a standard curve to derive input DNA content.
If a chemiluminescent hybridization detection tag is used and a reagent, such as an alkaline phosphatase substrate, is added to react with the detection tag, emitted photons will be measured instead of OD. This approach enhances the linear range of the measurements in that it avoids the loss of sensitivity in OD measurements experienced at high OD values. A suitable microplate reader for use with a chemiluminescent tag is commercially available from Dynatech of Chantilly, Va. Where a fluorescent tag is used, a suitable ELISA microplate reader is commercially available from Millipore of Boston, Mass.
B. Generation of Standard Curves for Calculation of the Ratio of Target to Competitor Nucleic Acid
Probe hybridization-based quantification of PCR products can eliminate false positive results derived from non-specific amplification. However, potential flaws can come from differences in hybridization or labeling efficiency of the probes. An exemplary construct for this purpose has tandemly arranged wild type DNA and competitor DNA sequences. Since the standard curves are generated from the results of hybridization of each probe with the standard construct, labeling or hybridization efficiency does not affect the results.
A nucleic acid standard (hereafter referred to for convenience as “standard DNA” construct) is constructed according to means known in the art to include two tandemly aligned DNA regions from wild type (target) DNA and competitor DNA. Conventional PCR is performed to amplify the standard DNA using a reactant modified primer (such as an aminated primer) and a regular primer. Two sets of serial dilutions of the standard DNA PCR products are prepared.
Separately, samples containing target DNA to which competitor DNA was added in known quantity are coamplified. Two aliquots of the PCR products are made and added to microtiter plate wells for covalent coupling of antisense strands and removal of sense strands. Hybridization is performed separately with the two sets of standard DNA solutions and the two sample/competitor DNA aliquots. The SSO's correspond to the target sequence and the competitor sequence. After hybridization, an assay appropriate to the detection tag used is performed and optical density or another appropriate value is measured with appropriate hybridization detection means.
Where the SSO's are not known to have equal hybridization efficiencies, a separate standard curve is generated for each SSO based on the adsorbence (OD) readings provided by use of the microtiter plate reader. (Only one curve is needed where no differences in the hybridization efficiencies for each probe is expected). ELISA data analysis software (such as the DELTA SOFT version 2.1 sold by BioMetallics of Princeton, N.J.) is then used to calculate the amount of target DNA in the sample. Using this data, a ratio of target to competitor products can be calculated.
Where the approximate amount of target DNA present in the sample is not known, target DNA samples can be mixed with various known concentrations (usually three) of competitor DNA. Competitive PCR is then performed according to the methods described herein and the ratio of target DNA to competitor calculated. A graph is then generated which plots the known concentrations of competitor against the ratios of target and competitor sequences determined by covalent capture PCR in logarithmic scales. Calculation of the amount of competitor which would give a 1:1 ratio will provide the approximate concentration of target DNA in the starting samples.
The following protocol is adapted from Diehl, et al., supra, and can be adapted for use with available equipment and reagents by those of ordinary skill in the art.
Prepare emulsifier-oil mix using 7% (wt/vol) ABIL WE09™, 20% (vol/vol) mineral oil and 73% (vol/vol) Tegosoft™ DEC. Vortex this mix briefly and incubate at 18-25° C. for 30 min. Store the mixture at room temperature for no longer than 2 d. Dilute the template DNA with TE to ˜20 μM immediately before use. DNA at low concentrations can stick to tubes during storage.
Set up a 150 μl amplification reaction by mixing the following: Primer 5 (2.5 μM) 3 μl, Primer 6 (400 μM) 3 μl, Template DNA (˜20 μM) 10 μl, Beads 6 μl, dNTPs mix 3 μl, 10×PCR buffer 15 μl, Platinum Taq DNA polymerase (5 U/μl) 9 μl, Water 101 μl.
Add, in order, one steel bead, 600 μl oil-emulsifier mix (Step 9) and 150 μl PCR mix to one well of a 96-well storage plate. Seal plate with adhesive film. Turn the plate upside down to make sure the steel bead moves freely in the well. Avoid excess oil on the rims of the wells as the adhesive film will not seal.
Purchase or assemble a TissueLyser™ adaptor set by sandwiching the 96-well storage plate containing the emulsion PCR mix between the top and bottom adapter plates, each fitted with a compression pad facing the 96-well storage plate. Place the assembly into the TissueLyser holder, and close the handles tightly. When using less than 192 wells, balance TissueLyser with a second adaptor set of the same weight. Mix once for 10 s at 15 Hz and once for 7 s at 17 Hz.
Disassemble the adaptor set and centrifuge the plate for 10 s at ˜3 g to get the liquid to the bottom. Assess the quality of the emulsions at 400× magnification with an inverted microscope. Dip a pipette tip into the emulsion, and streak it over the bottom of a 48-well cell culture plate. Aliquot 80 μl of the emulsion into eight wells of a 96 well PCR plate. Centrifuge the plate for 10 s at ˜3 g to get the liquid to the bottom.
Pipette emulsions slowly to avoid shear force. Temperature cycle the emulsions according to the following program: 2 min at 94° C., 2-4 15 s at 98° C., 45 s at 64° C., 75 s at 72° C., 15 s at 98° C., 45 s at 61° C., 75 s at 72° C., 15 s at 98° C., 45 s at 58° C., 75 s at 72° C., 15 s at 98° C., 45 s at 57° C., and 75 s at 72° C.
To each 80 μl emulsion, add 150 μl of Breaking buffer and pipette up and down three times to mix. Seal the PCR plate, place it into an empty 96-well storage plate, and assemble this between two TissueLyser™ adaptor plates as described. Place in TissueLyser™ and mix for 30 s at 20 Hz. Remove PCR plate from the TissueLyser and centrifuge for 2 min at 3,200 g. Remove the top oil layer with a 20 μl pipette tip attached to a vacuum manifold. Add 150 μl of Breaking buffer, seal the plate and centrifuge again for 2 min at 3,200 g. Place the plate in a 96-well magnetic separator for 1 min, and completely remove the liquid with a pipette.
Remove the plate from the magnet, resuspend the beads in 100 μl of TK buffer and pool the beads from the eight wells into a 1.5 ml tube. Place the tube on the magnet to concentrate the beads for 1 min, and carefully remove the supernatant with a pipette tip. This removes the non-biotinylated DNA strand from the beads. Resuspend the beads in 100 μl of TK buffer. The recovery of beads can be assessed by measuring absorption at 600 nm. A spectrophotometer is convenient for this purpose. An aliquot of the beads coated with Primer 5 can be used as a fiducial. The typical recovery with the procedure described is 50-70%.
Set up the oligohybridization in a 96-well PCR plate by mixing the following: Primer 3 (1 μM) 10 μl, Beads 20 μl, 5× hybridization buffer 20 μl, Water 50 μl.
The amount of beads to be used depends on the nature of the experiment. Ten million beads provide a great enough mass to be seen during magnetic collection and facilitate recovery. The recovery can be assessed by measuring absorption at 600 nm as described above.
To break emulsions and detect DNA on the beads, incubate the reaction at 50° C. for 15 min in a thermal a cycler. Place the plate on a 96-well magnetic separator for 1 min to concentrate the beads, and remove 80 μl of the supernatant with a pipette. Wash beads twice with 80 μl of TK buffer. Use flow cytometry to determine the relative fluorescence intensity of the primers hybridized to the DNA on the beads. Alternatively, fluorescence microscopy can provide a rapid qualitative analysis of the beads generated. Empirically, establish the amplifier gain (voltages) for the detection of the forward scatter and side scatter.
Once the primary multiplex PCR of the invention has identified deletion breakpoints in the target polynucleotide, it is possible to design primers to target the deleted sequence in the wild-type molecule. A pair of primers external to the deletion segment and a primer (“poison” primer) internal to it are utilized (see, schematic in
As described in Edgley, et al., Nuc. Acids Research, 30:e52, 2002, amplification from the wild type template produces two fragments, one full length and the other shorter. The latter will generally be produced more efficiently. Amplification from the variant template (lacking the deletion segment) produces a single variant fragment from the external primers. In a further PCR reaction, a primer pair is placed just inside the external first round primers.
The shorter wild-type band from the first round can't serve as a template for the second round because it lacks a primer binding site. The longer wild-type fragment does serve as a template but, because its production was limited in the first round of PCR, its production in the second round is also limited; i.e., was “poisoned” by the primer corresponding to the deleted segment.
The PAMP method was performed as described in Examples I and II. The total genomic DNA for each assay was 100 ng. This is equivalent to about 28000 copies of haploid genome (based on the estimate of 2.8×105 molecules/ug of haploid genome). The CDKN2A deleted cell line Detroit 562 was serially diluted with CDKN2A wild type HEK293 as shown in Table 1, below.
The assay was able to detect about 1 breakpoint sequence in the presence of 2000 fold excess of wild type genome with sensitivity of detecting 5-16 such molecules. Thus, the invention provides a method for detecting genomic DNA deletions in the presence of an excess of wild-type DNA; e.g., more than 99.9% wild-type.
Most of the TMPRSS2:ERG fusion junctions are between exons 1 or 2 of the TMPRSS2 and exons 2-5 of the ERG. Such constraints perhaps are related to whether a functional ERG protein can be made from the gene fusions. Therefore, a pair of primers at exon 1 of the TMPRSS and exon 6 of the ERG for RT-PCR were used.
As shown in
A prostate cancer cell line, VCaP, with a TMPRSS and ERG fusion was used for initial feasibility testing. The RT-PCR reaction was performed with an OneStep RT-PCR kit (Qiagen, Valencia, Calif.) essentially following the manufacturer's protocol, except that the final reaction volume was scaled down to 20 μl. The forward (GTT TCC CAG TCA CGA TCC AGG AGG CGG AGG GGG A) and reverse primers (GTT TCC CAG TCA CGA TCG GCG TTG TAG CTG GGG GTG AG) are located at exon 6 of ERG and exon 1 of TMPRSS2 respectively. The 5′ ends of both primer have the sequence of primer B (GTT TCC CAG TCA CGA TC) for the subsequent step of PCR labeling with a single primer B.
Briefly, the RT-PCR reaction was assembled at 4° C. in a PCR workstation and transferred to a thermocycler with the block preheated to 50° C. for 30 minutes and followed by 95° C. for 15 minutes to activate HotStar™ Taq DNA polymerase as well as to inactivate the reverse transcriptases. The PCR conditions were 35 cycles at 92° C. for 30 seconds, 55° C. for 30 seconds and 68° C. for 1.5 minutes with a final extension step at 68° C. for 5 minutes. One μl of unpurified product was subsequently used as a template for another 20 cycles of amplification to label the amplicons via a “Round C” PCR protocol (94° C. for 30 seconds, 40° C. for 30 seconds, 50° C. for seconds and 72° C. for 1 minute) with primer B and 4:1 mixture of aminoallyl dUTP (Ambion, Austin, Tex.) and dTTP for probe labeling (20). The labeled amplicons were purified with DNA Clean-up and Concentrator-5 columns (Zymo Research, CA). eluted in 9 μl of sodium bicarbonate (pH 9.0) and couple with 1 μl of DMSO dissolved Cy3 NHS esters (GE Healthcare, Piscataway, N.J.) for 30 to 60 minutes. The Cy3 labeled amplicons were purified with DNA Clean-up and Concentrator-5 columns and eluted with 10 μl of 10 mM Tris-HCL (pH 8.0). Then, the Cy3 labeled amplicons were diluted in water and combined with 3.6 μl of 20×SSC, 0.5 μl of Hepes (pH 7.0) and finally 0.5 μl of 10% SDS to reach final volume of 25 μl.
The total RNA was subjected to RT-PCR with a pair of primers located at exon 6 of ERG and exon 1 of TMPRSS2. The unpurified product was labeled and hybridized on the microarray (
To mimic a typical clinical situation, in which small population of cancer cells are present among normal host cells in a primary tumor, decreasing amounts of total RNA extracted from VCaP cells were spiked into an excess of HeLa RNA, which does not have the fusion transcripts. The detection limit reached by this particular assay was 32 pg of VCaP RNA in the presence of 100 ng of HeLa RNA (
To test the ability of the exon mapping array to detect and characterize TMPRSS2:ERG fusion transcripts in clinical samples, total RNA was isolated from frozen unpurified primary prostate tissues obtained during surgery. Many of these tumors had a substantial fraction of normal stromal cells. Total RNA (5-50 ng) from prostate cancers (n=20) and non-malignant hyperplastic prostate tissues (n=10) were subjected to RT-PCR labeling and array hybridization. The results showed that 7 of 20 cancer but zero of 10 non-malignant samples had TMPRSS2:ERG fusion genes.
To confirm the presence of the gene fusion, direct sequencing was performed for the 7 TMPRSS2:ERG positive samples. The sequencing data validated the exon fusion findings revealed by the array assay. Some samples clearly showed two or more bands on the agarose gel when the PCR products were subjected to electrophoresis, corresponding to two or more fusion transcripts in the same specimens. Therefore, multiple fusion transcripts may be detected in a single sample. The results are shown in Table 3:
Consistent with the VCaP titration study (
The invention having been fully described, modifications, equivalents and extensions thereof may become obvious to those of skill in the art in view of this disclosure. All such modifications, equivalents and extensions are considered to be within the scope of the invention and appended claims.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US07/17128 | 7/31/2007 | WO | 00 | 11/17/2009 |
| Number | Date | Country | |
|---|---|---|---|
| 60834684 | Jul 2006 | US |
