US20020058265A1 - Detection of microsatellite instability and its use in diagnosis of tumors - Google Patents

Detection of microsatellite instability and its use in diagnosis of tumors Download PDF

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US20020058265A1
US20020058265A1 US09/841,366 US84136601A US2002058265A1 US 20020058265 A1 US20020058265 A1 US 20020058265A1 US 84136601 A US84136601 A US 84136601A US 2002058265 A1 US2002058265 A1 US 2002058265A1
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seq
locus
bat
loci
mono
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Jeffery Bacher
Laura Flanagan
Nadine Nassif
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Promega Corp
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Priority to AT02731467T priority patent/ATE454468T1/de
Priority to ES02731467T priority patent/ES2338765T3/es
Priority to JP2002583932A priority patent/JP2004533241A/ja
Priority to EP02731467A priority patent/EP1390538B1/fr
Priority to PCT/US2002/012779 priority patent/WO2002086448A2/fr
Priority to DE60235005T priority patent/DE60235005D1/de
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Publication of US20020058265A1 publication Critical patent/US20020058265A1/en
Priority to US10/314,810 priority patent/US7202031B2/en
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • This invention relates to the detection of instability in regions of genomic DNA containing simple tandem repeats, such as microsatellite loci.
  • the invention particularly relates to multiplex analysis for the presence or absence of instability in a set of microsatellite loci in genomic DNA from cells, tissue, or bodily fluids originating from a tumor.
  • the invention also relates to the use of microsatellite instability analysis in the detection and diagnosis of cancer and predisposition for cancer.
  • Microsatellite loci of genomic DNA have been analyzed for a wide variety of applications, including, but not limited to, paternity testing, forensics work, and in the detection and diagnosis of cancer. Cancer can be detected or diagnosed based upon the presence of instability at particular microsatellite loci that are unstable in one or more types of tumor cells.
  • a microsatellite locus is a region of genomic DNA with simple tandem repeats that are repetitive units of one to five base pairs in length. Hundreds of thousands of such microsatellite loci are dispersed throughout the human genome. Microsatellite loci are classified based on the length of the smallest repetitive unit. For example, loci with repetitive units of 1 to 5 base pairs in length are termed “mononucleotide”, “dinucleotide”, “trinucleotide”, “tetranucleotide”, and “pentanucleotide” repeat loci, respectively.
  • Each microsatellite locus of normal genomic DNA for most diploid species, such as genomic DNA from mammalian species, consists of two alleles at each locus.
  • the two alleles can be the same or different from one another in length and can vary from one individual to the next.
  • Microsatellite alleles are normally maintained at constant length in a given individual and its descendants; but, instability in the length of microsatellites has been observed in some tumor types (Aaltonen et al., 1993, Science 260:812-815; Thibodeau et al.,1993 Science 260:816-819; Peltomaki et al., 1993 Cancer Research 53:5853-5855; Ionov et al., 1993 Nature 363:558-561).
  • MSI microsatellite instability
  • HNPCC Hereditary Nonopolyposis Colorectal Cancer
  • MSI in HNPCC is thought to be a dysfunctional DNA mismatch repair system that fails to reverse errors that occur during DNA replication (Fishel et al., 1993 Cell 75:1027-38; Leach et al., 1993 Cell 75:215-25; Bronner et al., 1994 Nature 368:258-61; Nicolaides et al., 1994 Nature 371:75-80; Miyaki et al., 1997 Nat Genetics 17:271-2).
  • Insertion or deletion of one or more repetitive units during DNA replication persists without mismatch repair and can be detected as length polymorphisms by comparison of allele sizes found in microsatellite loci amplified from normal and tumor DNA samples (Thibodeau et al., 1993, supra).
  • MSI has been found in over 90% of HNPCC and in 10-20% of sporadic colorectal tumors (Liu et al., 1996 Nature Med 2:169-174; Thibodeau et al., 1993, supra; Ionov et al., 1993 Nature 363:558-561; Aaltonen et al., 1993 Science 260: 812-816; Lothe et al., 1993 Cancer Res. 53: 5849-5852). However, MSI is not limited to colorectal tumors.
  • MSI has also been detected in pancreatic cancer (Han et al., 1993 Cancer Res 53:5087-5089) gastric cancer (Id.; Peltomaki et al., 1993 Cancer Res 53:5853-5855; Mironov et al., 1994 Cancer Res 54:41-44; Rhyu et al., 1994 Oncogene 9:29-32; Chong et al., 1994 Cancer Res 54:4595-4597), prostate cancer (Gao et al., 1994 Oncogene 9:2999-3003), endometrial cancer (Risinger et al., 1993 Cancer Res 53:5100-5103; Peltomaki et al., 1993 Cancer Res 53:5853-5855), and breast cancer (Patel et al., 1994 Oncogene 9:3695-3700).
  • HNPCC DNA mismatch repair genes
  • MSI-H high frequency MSI
  • MSH6, MSH3, PMS1 and PMS2 genes have been reported in HNPCC patients, indicating that inherited mutations in these mismatch repair genes play a minor role in HNPCC (Peltomaki et al., 1997 Gastroenterologly 113:1146-1158; Liu et al., 1996 Nat Med 2:169-174; Kolodner et al., 1999 Cancer Research 59:5068-5074). Without functional repair proteins, errors that occur during replication are not repaired leading to high mutation rates and increased likelihood of tumor development.
  • MSI positive tumors have been found to carry somatic frameshift mutations in mononucleotide repeats in the coding region of several genes involved in growth control, apoptosis, and DNA repair (e.g., TGFBRII, BAX, IGFIIR, TCF4, MSH3, MSH6) (Planck et al., 2000 Genes, Chromosomes & Cancer 29:33-39; Yamamoto et al., 1998 Cancer Research 58:997-1003; Grady et al., 1999 Cancer Research 59:320-324; Markowitz et al., 1995 Science 268:1336-1338; Parsons et al., 1995 Cancer Research 55:5548-5550).
  • TGFBRII The most commonly altered locus is TGFBRII, in which over 90% of MSI-H colon tumors have been found to contain a mutation in the 10 base polyadenine repeat present in the gene (Markowitz et al., 1995 Science 268:1336-1338).
  • MSI occurs in almost all HNPCC tumors regardless of which mismatch repair gene is involved. MSI has also been shown to occur early in tumorigenesis. These two factors contribute to making MSI analysis an excellent diagnostic test for the detection of HNPCC. In addition, MSI analysis can serve as a useful pre-screening test to identify potential HNPCC patients for further genetic testing. MSI analysis of sporadic colorectal carcinomas is also desirable, since the occurrence of MSI correlates with a better prognosis (Bertario et al., 1999 International J Cancer 80:83-7).
  • DNA analysis of microsatellite loci makes it theoretically possible to develop a blood test for use in the detection of specific types of cancer.
  • Early studies have shown that tumor DNA is released into the circulation, and is present in particularly high concentrations in plasma and serum in a number of different types of cancer (Leon et al., 1977 Cancer Res 37:646-650; Stroun et al., 1989 Oncology 46:318-322).
  • PCR polymerase chain reaction
  • the first tumor-specific gene sequences detected in blood from patients with cancer were mutated K-ras genes (Vasioukhin et al., 1994 Br. J Haematol 86: 774-779; Sorenson et al., 1994 Cancer Epidemiol. Biomark. Prev. 3:67-71; Sorenson et al., 2000 Clinical Cancer Research 6:2129-2137; Anker et al., 1997 Gastroenterology 112:1114-1120).
  • Hibi et al. also reported that eighty percent of primary tumors in the colon cancer patients displayed MSI and/or loss of heterozygosity (hereinafter, “LOH”), another type of mutation discussed below. No microsatellite or LOH mutations were detected in paired serum DNA. However, identical K-ras mutations were found in corresponding tumor and serum DNAs, indicating that tumor DNA was present in the blood. (Id.)
  • circulating tumor cells and micrometastases may also have important prognostic and therapeutic implications. Because disseminated tumor cells are present in very small numbers, they are not easily detected by conventional immunocytological tests, which can only detect a single tumor cell among 10,000 to 100,000 normal cells (Ghoussein et al., 1999 Clinical Cancer Research 5:1950-1960). More sensitive molecular techniques based on PCR amplification of tumor-specific abnormalities in DNA or RNA have greatly facilitated detection of occult (hidden) tumor cells.
  • PCR-based tests capable of routinely detecting one tumor cell in one million normal cells have been devised for identification of circulating tumor cells and micrometastases in leukemias, lymphomas, melanoma, neuroblastoma, and various types of carcinomas. (Id.)
  • the frequency of MSI observed with a particular tumor type in a single study will depend on the number of tumors analyzed, the number of loci investigated, how many loci need to be altered to score a tumor as having MSI and which particular loci were included in the analysis.
  • the National Cancer Institute sponsored a workshop on MSI to review and unify the field (Id.).
  • a panel of five microsatellites was recommended as a reference panel for future research in the field. This panel included two mononucleotide loci BAT-25, BAT-26, and three dinucleotide loci D5S346, D2S123, D17S250.
  • Clinical diagnostic assays used for determining treatment and prognosis of disease require that the tests be highly accurate (low false negatives) and specific (low false positive rate).
  • Many informative microsatellite loci have been identified and recommended for MSI testing (Boland et al. 1998, supra). However, even the most informative microsatellite loci are not 100% sensitive and 100% specific.
  • multiple markers can be used to increase the power of detection. The increased effort required to analyze multiple markers can be offset by multiplexing. Multiplexing allows simultaneous amplification and analysis of a set of loci in a single tube and can often reduce the total amount of DNA required for complete analysis.
  • MSI analysis solves problems of accuracy and discrimination of MSI phenotypes, but the additional complexity can make analysis more challenging. For example, when microsatellite loci are co-amplified and analyzed in a multiplex format, factors affecting ease and accuracy of data interpretation become much more essential. One of the primary factors affecting accurate data interpretation is the amount of stutter that occurs at microsatellite loci during PCR (Bacher & Schumm, 1998 Profiles in DNA 2:3-6; Perucho, 1999 Cancer Research 59:249-256). Stutter products are minor fragments produced by the PCR process that differ in size from the major allele by multiples of the core repeat unit.
  • microsatellite loci tends to be inversely correlated with the length of the core repeat unit. Thus, stutter is most severely displayed with mono- and dinucleotide repeat loci, and to a lesser degree with tri-, tetra-, and pentanucleotide repeats (Bacher & Schumm, 1998, supra). Use of low stutter loci in multiplexes would greatly reduce this problem. However, careful selection of loci is still necessary in choosing low stutter loci because percent stutter can vary considerably even within a particular repeat type (Micka et al., 1999 Journal of Forensic Sciences 44:1-15).
  • Microsatellite multiplex systems have been primarily developed for use in genotyping, mapping studies and DNA typing applications. These multiplex systems are designed to allow co-amplification of multiple microsatellite loci in a single reaction, followed by detection of the size of the resulting amplified alleles.
  • Matching probability is a common statistic used in DNA typing that defines the number of individuals you would have to survey before you would find the same DNA pattern as a randomly selected individual.
  • a four locus multiplex system (GenePrintTM CTTv Multiplex System, Promega) has a matching probability of 1 in 252.4 in African-American populations, compared to an eight locus multiplex system (GenePrintTM PowerPlexTM 1.2 System, Promega) which has a matching probability of 1 in 2.74 ⁇ 10 8 ( Proceedings: American Academy of Forensic Sciences (Feb. 9-14, 1998), Schumm, James W. et al., p. 53, B88; Id. Gibson, Sandra D. et al., p. 53, B89; Id., Lazaruk, Katherine et al., p. 51, B83; Sparkes, R.
  • the materials and methods of the present invention are designed for use in multiplex analysis of particular microsatellite loci of human genomic DNA from various sources, including various types of tissue, cells, and bodily fluids.
  • the present invention represents a significant improvement over existing technology, bringing increased power of discrimination, precision, and throughput to the analysis of MSI loci and to the diagnosis of illness, such as cancer, related to mutations at such loci.
  • the present invention provides methods and kits for amplifying and analyzing microsatellite loci or sets of microsatellite loci.
  • the present invention also provides methods and kits for detecting cancer in an individual by co-amplifying multiple microsatellite loci of human genomic DNA originating from tumor tissue or cancerous cells.
  • the present invention provides a method of analyzing micro-satellite loci, comprising: (a) providing primers for co-amplifying in a single tube a set of at least three microsatellite loci of genomic DNA, comprising at least one mononucleotide repeat locus and at least two tetranucleotide repeat loci; (b) co-amplifying the set of at least three microsatellite loci from a sample of genomic DNA in a multiplex amplification reaction, using the primers, thereby producing amplified DNA fragments; and (c) determining the size of the amplified DNA fragments.
  • the present invention provides a method of co-amplifying the set of at least three microsatellite loci of at least two different samples of genomic DNA, a first sample originating from normal non-cancerous biological material from an individual and a second sample originating from a second biological material from the individual.
  • the at least two samples of human genomic DNA are co-amplified in separate multiplex amplification reactions, using primers to each of the loci in the set of at least three microsatellite loci.
  • the size of the resulting amplified DNA fragments from the two multiplex reactions are compared to one another to detect instability in any of the at least three microsatellite loci of the second sample of human genomic DNA.
  • Another embodiment of the present invention is a method of analyzing at least one mononucleotide repeat locus of human genomic DNA selected from the group consisting of MONO-11 and MONO-15.
  • the method of analyzing the at least one mononucleotide repeat locus selected from the group consisting of MONO-11 and MONO-15 comprises the steps of: (a) providing at least one primer of the at least one mononucleotide repeat locus; (b) amplifying the at least one mononucleotide repeat locus from a sample of genomic DNA originating from a biological material from an individual human subject, using the at least one primer, thereby producing an amplified DNA fragment; and (c) determining the size of the amplified DNA fragments.
  • the amplified DNA fragments are preferably analyzed to detect microsatellite instability at the at least one mononucleotide repeat locus by comparing the size of the amplified DNA fragments to the most commonly observed allele size at that locus in a human population.
  • the method is used to amplify the at least one mononucleotide repeat locus of a sample of human genomic DNA from normal non-cancerous biological material from an individual, and microsatellite instability is detected by comparing the resulting amplified DNA fragments to those obtained in step (b).
  • Another embodiment of the present invention is a kit for the detection of microsatellite instability in DNA isolated from an individual subject, comprising a single container with oligonucleotide primers for co-amplifying a set of at least three microsatellite loci comprising one mononucleotide locus and two tetranucleotide loci.
  • the various embodiments of the method and kit of the present invention are particularly suited for use in the detection of MSI in tumor cells or cancerous cells.
  • the method or kit of the present invention can be used to amplify at least one mononucleotide repeat locus selected from the group consisting of MONO-11 and MONO-15 or the set of at least three microsatellite loci comprising at least one mononucleotide repeat locus and at least two tetranucleotide repeat loci of at least one sample of genomic DNA from biological material, such as tissue or bodily fluids, preferably biological material containing or suspected of containing DNA from tumors or cancerous cells.
  • FIG. 1 shows a tetranucleotide repeat (GATA), amplified by a primer pair (“primer A” and “primer B”) in a polymerase chain reaction (“PCR”), followed by separation of amplified alleles by size using capillary electrophoresis, and a plot of the fractionated amplified alleles using GeneScanTM software. Note that only the two alleles and small stutter peaks appear in the plot of amplified DNA from normal DNA, while three MSI peaks appear in addition to the two allele peaks in the plot of amplified tumor DNA.
  • GATA tetranucleotide repeat
  • FIG. 1 Illustration of microsatellite instability analysis.
  • the figure is a diagram of a primer pair annealed to a tetranucleotide locus on two alleles of the same genomic DNA, and plots of results of capillary electrophoresis of products of amplification of a tetranucleotide locus of DNA originating from normal vs. tumor tissue. MSI peaks are indicated in the plot of amplified DNA from tumor tissue.
  • FIG. 2 Illustration of effect of microsatellite repeat unit length on amount of stutter observed.
  • the figure includes a diagram of a primer pair annealed to a tetranucleotide repeat locus on two different alleles of genomic DNA, and a set of fluorescent scans and plots of amplified mono-, di-, tri-, tetra-, and pentanucleotide repeat loci from human genomic DNA from various individuals, amplified and fractionated by gel or by capillary electrophoresis.
  • FIG. 3 Demonstration that low stutter tetranucelotide repeat loci are easier to interpret than high stutter dinucleotide repeat loci.
  • the figure is a plot of results of capillary electrophoresis of products of the amplification of two tetranucleotide and two dinucleotide repeat loci of two different sets of samples of DNA originating from normal vs. tumor tissue.
  • FIG. 4 Illustration of variance in amount of stutter within selected tetranucleotide and pentanucleotide repeat loci.
  • the figure is a plot of the variability in percent stutter observed in a 13 different tetranucleotide and 5 different pentanucleotide repeat loci.
  • the boxes represent the average percent stutter and the solid bars the range of stutter observed for each locus.
  • FIG. 5 Results of screening of tetranucleotide repeat markers for frequency of microsatellite instability.
  • the figure is a plot of the number of microsatellite loci, out of a total of 273 markers, that displays a given percent MSI. For example, approximately 15 loci were altered in 100% of MSI-H tumor samples evaluated.
  • FIG. 6 Results of screening of pentanucleotide repeat markers for frequency of microsatellite instability.
  • the figure is a plot of the percent MSI observed for each of eight different tetranucleotide repeat loci in a set of nine MSI-H and a set of 30 MSS tumors.
  • FIG. 7 Microsatellite instability analysis using MONO-15 marker.
  • the figure is a plot generated from capillary electrophoresis products of amplification of the MONO-15 locus of DNA from four different sets of paired normal and tumor samples originating from four different individuals.
  • FIG. 8 Percent MSI in 59 colon cancer samples using nine-locus MSI multiplex.
  • the figure is a plot of the percent MSI observed in 59 colon cancer samples (29 MSH and 30 MSI-L or MSS samples) using the nine locus MSI multiplex described in Example 6, below (i.e., D1S518, D3S2432, D7S1808, D7S3046, D7S9070, D10S1426, BAT-25, BAT-26, and MONO-15).
  • FIG. 9 Fluorescent multiplex microsatellite analysis using a nine-locus MSI Multiplex.
  • the figure is a plot generated from capillary electrophoresis of products of multiplex amplification of normal non-cancerous human genomic, using the nine locus MSI multiplex used in FIG. 8.
  • FIG. 10 Detection of microsatellite instability in colon cancer samples using a nine-locus MSI multiplex.
  • the figure is a plot generated from capillary electrophoresis of products of multiplex amplification of DNA from paired normal and colon tumor sample, using the nine locus MSI multiplex used in FIG. 8.
  • FIG. 11 Detection of microsatellite instability in colon cancer samples using nine-locus MSI multiplex is the same type of plot shown in FIG. 10, generated using a different sample of paired normal and colon cancer DNA from a different individual.
  • FIG. 12 Detection of microsatellite instability in stomach cancer samples using nine-locus MSI multiplex.
  • the figure is a plot generated from capillary electrophoresis of products of multiplex amplification of DNA from paired normal and stomach cancer tumor samples, using the nine locus MSI multiplex described in FIG. 8.
  • FIG. 13 Microsatellite analysis of paraffin embedded tissues with nine-locus MSI multiplex. The figure is a plot generated from capillary electrophoresis of products of multiplex amplification of DNA from paraffin embedded tissue, using the nine locus MSI multiplex described in FIG. 8.
  • FIG. 14 Percent MSI in colon cancer samples at 10 different microsatellite loci.
  • the figure is a bar graph of the percent MSI observed in 66 colon cancer samples (36 MSI-H and 30 MSI stable or MSI-L samples) using a nine loci contained in the MSI multiplex described in Example 8 (i.e., BAT-26, D7S3070, D7S3046, BAT-40, MONO-15, D7S1808, BAT-25, D10S1426 and D3S2432) and D1S518.
  • FIG. 15 Fluorescent multiplex microsatellite analysis using a nine-locus MSI Multiplex.
  • the figure is a plot generated from capillary electrophoresis of products of multiplex amplification of normal non-cancerous human genomic DNA, using the nine-locus MSI multiplex described in Example 8 labeled with primers labeled with fluorescent dyes, as follows. Primers to BAT-26, D7S3070, and D7S3046 were labeled with fluorescein; primers to BAT-40, MONO-15 and D7S1808 were labeled with JOE; and primers to BAT-25, D10S1426 and D3S2432 were labeled with TMR.
  • FIG. 16 Detection of microsatellite instability in colon cancer samples using a nine-locus MSI multiplex.
  • the figure is a plot generated from capillary electrophoresis of products of multiplex amplification of DNA from paired normal and colon tumor samples, using the same nine locus MSI multiplex and labeled primers used in FIG. 15.
  • Allele refers to one of several alternative forms of a gene or DNA sequence at a specific chromosomal location (locus). At each autosomal locus an individual possesses two alleles, one inherited from the father and one from the mother.
  • “Amplify”, as used herein, refers to a process whereby multiple copies are made of one particular locus of a nucleic acid, such as genomic DNA. Amplification can be accomplished using any one of a number of known means, including but not limited to the polymerase chain reaction (PCR) (Saiki, R. K., et al., 1985 Science 230: 1350-1354), transcription based amplification (Kwoh, D. Y., and Kwoh, T. J., American Biotechnology Laboratory, October, 1990) and strand displacement amplification (SDA) (Walker, G. T., et al., 1992 Proc. Natl. Acad. Sci., U.S.A. 89: 392-396).
  • PCR polymerase chain reaction
  • SDA strand displacement amplification
  • Co-amplify refers to a process whereby multiple copies are made of two or more loci in the same container, in a single amplification reaction.
  • DNA polymorphism refers to the existence of two or more alleles for a given locus in the population.
  • Locus or “genetic locus”, as used herein, refers to a unique chromosomal location defining the position of an individual gene or DNA sequence.
  • Locus-specific primer refers to a primer that specifically hybridizes with a portion of the stated locus or its complementary strand, at least for one allele of the locus, and does not hybridize efficiently with other DNA sequences under the conditions used in the amplification method.
  • LOH Loss of Heterozygosity
  • “Microsatellite Locus”, as used herein, refers to a region of genomic DNA that contains short, repetitive sequence elements of one (1) to seven (7), more preferably one (1) to five (5), most preferably one (1) to four (4) base pairs in length. Each sequence repeated at least once within a microsatellite locus is referred to herein as a “repeat unit.” Each microsatellite locus preferably includes at least seven repeat units, more preferably at least ten repeat units, and most preferably at least twenty repeat units.
  • MSI microsatellite Instability
  • MSI refers to a form of genetic instability in which alleles of genomic DNA obtained from certain tissue, cells, or bodily fluids of a given subject change in length at a microsatellite locus.
  • MSI can be observed upon amplification of two different samples of genomic DNA from a particular subject, such as DNA from healthy and cancerous tissue, wherein the normal sample produces amplified alleles of one or two different lengths and the tumor sample produces amplified alleles wherein at least one of the alleles is of a different length from the amplified alleles of the normal sample of DNA at that locus.
  • MSI generally appears as a result of the insertion or deletion of at least one repeat unit at a microsatellite locus.
  • MSI-H is a term used to classify tumors as having a high frequency of MSI.
  • five microsatellite loci are analyzed, such as the five microsatellite loci of selected by a workshop on HNPCC at the National Cancer Institute in 1998 for use in the detection of HNPCC, a tumor is classified as MSI-H when at least two of the loci show instability (Boland, 1998 Cancer Research 58: 5248-5257).
  • a tumor is classified as MSI-H when at least 30% of the microsatellite loci of genomic DNA originating from the tumor is are found to be unstable.
  • MSI-L is a term used to classify tumors as having a low frequency of MSI.
  • a tumor is classified as MSI-L when only one of the loci shows instability.
  • MSI-L is classified as MSI-L when less than 30% of the microsatellite loci of genomic DNA originating from the tumor is are found to be unstable.
  • MSI-L tumors are thought to represent a distinct mutator phenotype with potentially different molecular etiology than MSI-H tumors (Thibodeau, 1998; Wu et al., 1999, Am J Hum Genetics 65:1291-1298). To accurately distinguish MSI-H and MSI-L phenotypes it has been recommended that more than five microsatellite markers be analyzed (Boland, 1998, supra; ; Frazer et al., 1999 Oncology Research 6:497-505).
  • MSS refers to tumors which are microsatellite stable, when no microsatellite loci exhibit instability.
  • the distinction between MSI-L and MSS can also only be accomplished when a significantly greater number of markers than five are utilized.
  • National Cancer Institute recommended use of an additional 19 mono- and dinucleotide repeat loci for this purpose, and for the purpose of making clearer distinctions between MSI-H and MSI-L tumors, as described above (Boland, 1998, supra).
  • MSI-L/S refers to all classified as either MSI-L or MSS.
  • “Microsatellite marker”, as used herein, refers to a fragment of genomic DNA which includes a microsatellite repeat and nucleic acid sequences flanking the repeat region.
  • “Monomorphic”, as used herein, refers to a locus of genomic DNA where only one allele pattern has been found to be present in the normal genomic DNA of all members of a population.
  • Nucleotide refers to a basic unit of a DNA molecule, which includes one unit of a phosphatidyl back bone and one of four bases, adenine (“A”); thymine (“T”); guanine (“G”); and cytosine (“C”).
  • Polymerase chain reaction refers to a technique in which cycles of denaturation, annealing with primer, and extension with DNA polymerase are used to amplify the number of copies of a target DNA sequence by approximately 10 6 times or more.
  • the polymerase chain reaction process for amplifying nucleic acid is covered by U. S. Pat. Nos. 4,683,195 and 4,683,202, which are incorporated herein by reference for a description of the process.
  • Primer refers to a single-stranded oligonucleotide or DNA fragment which hybridizes with a strand of a locus of target DNA in such a manner that the 3′ terminus of the primer may act as a site of polymerization using a DNA polymerase enzyme.
  • Primer pair refers to a pair of primers which hybridize to opposite strands a target DNA molecule, to regions of the target DNA which flank a nucleotide sequence to be amplified.
  • Primer site refers to the area of the target DNA to which a primer hybridizes.
  • Quasi-monomorphic refers to a locus of genomic DNA where only one allele pattern has been found to be present in the normal genomic DNA of almost all the members of a population.
  • “Stutter”, as used herein, refers to a minor fragment observed after amplification of a microsatellite locus, one or more repeat unit lengths smaller than the predominant fragment or allele. It is believed to result from a DNA polymerase slippage event during the amplification process (Levinson & Gutman, 1987 Molecular Biology Evolution 4:203; Schlotterer and Tautz, 1992 Nucleic Acids Research 20:21 1).
  • At least one MSI locus amplified or co-amplified in each of the embodiments of the present invention illustrated and discussed herein is a mononucleotide repeat locus.
  • Such loci have been shown to be very susceptible to alteration in tumors with dysfunctional DNA mismatch repair systems (Parsons, 1995 supra), making such loci particularly useful for the detection of cancer and other diseases associated with dysfunctional DNA mismatch repair systems.
  • One group of researchers reported that by amplifying and analyzing a single mononucleotide repeat locus, BAT-26, they were able to correctly confirm the MSI-H status of 159 out of 160 (99.4% accuracy) tumor samples (Hoang et al., 1997 Cancer Research 57:300-303).
  • Some mononucleotide repeat loci including BAT-26, have also been identified as having quasi-monomorphic properties. Monomorphic or quasi-monomorphic properties make the comparison of normal/tumor pairs simpler, since PCR products from normal samples are generally all the same size and any alterations in tumor samples are easily identified.
  • a mononucleotide locus is monomorphic or quasi-monomorphic, however, one can readily detect shifts in the size of an allele, indicating MSI, even in the presence of a high degree of stutter.
  • detection of shifts in size can be done by comparison of amplified alleles from genomic DNA from biological material of an individual, such as tumor tissue or bodily fluids, suspected of exhibiting microsatellite instability to the most commonly observed allele size at that locus in a population. This feature enables one to use a single standard or panel of standard allele patterns to analyze individual results, minimizing the amount of samples which must be taken from an individual in order to detect microsatellite instability in certain genomic DNA of the individual.
  • At least one of the microsatellite loci amplified in the method or using the kit of the present invention is preferably a mononucleotide repeat locus, more preferably a quasi-monomorphic mononucleotide repeat locus.
  • the mononucleotide repeat locus selected for use in the methods and kits of the present invention is preferably unstable in cancerous biological material, but not in normal biological material.
  • BAT-25, BAT-26 and BAT-40 have been identified as mononucleotide repeat loci useful in the identification of MSI in colorectal tumors characteristic of Hereditary Nonpolyposis Colon Cancer (Zhou et al., 1998 Genes, Chromosomes & Cancer 21:101-107; Boland et al, 1998 Cancer Research 58:5248-5257, Dietmaier et al., 1997 Cancer Research 57:4749-4756; Hoang et al., 1997 Cancer Research 57:300-303).
  • Two additional loci, identified herein as MONO-11 and MONO- 15 were identified through a search of a public computerized database of sequence information (GenBank), and found to have the preferred characteristics for such loci, identified above.
  • the search for and identification of mononucleotide repeat loci suitable for use in the present invention is illustrated in Example 2. Similar techniques could be used to identify other mononucleotide repeat loci suitable for use in the methods and kits of the present invention.
  • the mononucleotide repeat loci amplified or co-amplified according to the present methods or using the present kits are preferably quasi-monomorphic and exhibit instability in the type of tissue of interest for a given application.
  • MONO-11 and MONO-15, BAT-25, and particularly BAT-26 are all quasi-monomorphic monomorphic and exhibit instability in several cancerous tumor tissues. All four quasi-monomorphic mononucleotide repeat loci have been found to be particularly useful in the methods and kits of the present invention.
  • BAT-40 has also been found to be useful in the methods and kits of the present invention, due to its instability in several cancerous tumor tissues. However, BAT-40 is not a quasi-monomorphic locus. At least one, more preferably at least two mononucleotide repeat microsatellite loci are amplified or co-amplified in the method of the present invention.
  • At least one mononucleotide repeat locus and at least two tetranucleotide repeat loci are co-amplified and analyzed according to at least some embodiments of the method and kits of the present invention.
  • Tetranucleotide repeat loci inherently generate very few stutter artifacts when amplified, compared to microsatellite loci with shorter repeat units, particularly compared to mono- and dinucleotide repeat loci. (See, e.g., FIG. 2.) Such artifacts can be difficult to distinguish from MSI if a shifted allele occurs at the stutter position of the second allele.
  • the tetranucleotide repeat loci are preferably selected on the basis of being stable in the DNA of an individual except in the type of biological material of interest.
  • Preferred tetranucleotide repeat loci used in the methods and kits of the present invention include: FGA, D1S518, D1S547, D1S1677, D2S1790, D3S2432, D5S818, D5S2849, D6S1053, D7S3046, D7S1808, D7S3070, D8S1179, D10S1426, D10S2470, D12S391, D17S1294, D17S1299, and D18S51.
  • Additional mononucleotide or tetranucleotide loci with the same preferred criteria described above are preferably co-amplified with the set of at least three microsatellite loci described above.
  • microsatellite loci other than mononucleotide repeat or tetranucleotide repeat loci could be included in the set of at least three microsatellite loci co-amplified and analyzed according to the method or using the kit of the present invention.
  • the method or kit of the present invention When the method or kit of the present invention is to be used in clinical diagnostic assays to be used to determine treatment and prognosis of disease, it must be designed to produce results which are highly accurate (low false negatives) and specific (low false positive rate).
  • Informative microsatellite loci are preferably identified by screening, more preferably by very extensive screening (see Examples 1 and 2). However, even the most informative microsatellite loci are not 100% sensitive and 100% specific.
  • the power of individual markers at detecting the presence of MSI in tissue associated with a particular disease, such as cancerous tumors, can be increased tremendously by multiplexing multiple markers. Increased information yielded from amplifying and analyzing greater numbers of loci results in increased confidence and accuracy in interpreting test results. To obtain needed sensitivity in detecting or diagnosing diseases such as cancer, it has been recommended that one analyze five or more highly informative microsatellite loci (Boland, 1998 Cancer Research 58: 5248-5257). Multiplexing of microsatellite loci further simplifies MSI analysis by allowing simultaneous amplification and analysis of all multiple loci, while reducing the amount of often-limited DNA required for amplification.
  • MSI determination Another common problem in MSI determination relates to the occurrence of an intermediate MSI phenotype where only a small percentage ( ⁇ 30%) of microsatellite markers are altered in tumors (Boland, 1998, supra). These MSI-low tumors are thought to represent a distinct mutator phenotype with potentially different molecular etiology than MSI-H tumors (Thibodeau et al, 1993 Science 260: 816-8; Wu et al., 1999 Am J Hum Genetics 65:1291-1298; Kolodner et al., 1999 Cancer Research 59:5068-5074; Wijnen et al., 1999 Nature Genetics 23:142-144 ).
  • MSI-L and MSS tumors are generally considered as one stable phenotypic class.
  • MSI-H and MSI-L phenotypes it has been recommended that multiple microsatellite markers be analyzed (Boland, 1998; Frazer, 1999 supra).
  • Tetranucleotide repeat loci were chosen for inclusion in the MSI multiplex analyzed according to the method and using the kit of the present invention because they display considerably less stutter that shorter repeat types like dinucleotides (FIG. 2). However, careful selection of loci is still necessary in choosing low stutter loci because % stutter can vary considerably even within a particular repeat type (FIG. 4). Mononucleotide repeat loci were chosen for individual analysis and for inclusion in the MSI Multiplex because of high rates of instability in diseased biological material of interest.
  • LOH incidence of LOH is another factor in the selection of MSI loci to be amplified and analyzed in the methods or kits of the present invention.
  • LOH can result in misidentification of a missing normal allele at a microsatellite marker as an indication of MSI when no other novel fragments are present for that marker. Specifically, one cannot easily discern whether this represents true LOH or MSI in which the shifted allele has co-migrated with the remaining wild-type allele.
  • the microsatellite markers selected for use in the present methods and kits preferably exhibit a low frequency of LOH, preferably no more than about 20% LOH, more preferably no more than about 14% LOH, even more preferably, no more than about 3% LOH.
  • microsatellite marker It is a relatively uncommon occurrence for a microsatellite marker to possess all necessary attributes described above (i.e., high sensitivity, high specificity, low stutter, low LOH).
  • the threshold for an MSI analysis system to be used in a diagnostic test is even higher, requiring robust and reproducible results from multiple loci in one assay using small quantities of DNA from difficult samples and be able to distinguish between MSI-L and MSI-H phenotypes. All the specific preferred mono- and tetranucleotide repeat loci identified herein above as being preferred for use in the present invention were found to meet each of the criteria for MSI loci suitable for use in diagnostic analysis, set forth herein above.
  • Primers for one or more microsatellite loci are provided in each embodiment of the method and kit of the present invention. At least one primer is provided for each locus, more preferably at least two primers for each locus, with at least two primers being in the form of a primer pair which flanks the locus.
  • the primers are to be used in a multiplex amplification reaction it is preferable to select primers and amplification conditions which generate amplified alleles from multiple co- amplified loci which do not overlap in size or, if they do overlap in size, are labeled in a way which enables one to differentiate between the overlapping alleles.
  • Primers suitable for the amplification of individual loci preferably co-amplified according to the methods of the present invention are provided in Example 4, Table 9, herein below. Primers suitable for use in one preferred multiplex of nine loci (i.e., BAT-25, D10S1426, D3S2432, BAT-26, D7S3046, D7S3070, MONO-15, D1S518, and D7S1808) are described in Example 6, Table 11. Primers suitable for use in a more preferred multiplex of nine loci (i.e., BAT-25, D10S1426, D3S2432, BAT-26, D7S3046, D7S3070, MONO-15, BAT-40, and D7S1808) are described in Example 8, Table 13. Guidance for designing these and other multiplexes is provided, below. It is contemplated that other primers suitable for amplifying the same loci or other sets of loci falling within the scope of the present invention could be determined by one of ordinary skill in the art.
  • the method of multiplex analysis of microsatellite loci of the present invention contemplates selecting an appropriate set of loci, primers, and amplification protocols to generate amplified alleles from multiple co-amplified loci which preferably do not overlap in size or, more preferably, which are labeled in a way which enables one to differentiate between the alleles from different loci which overlap in size. Combinations of loci may be rejected for either of the above two reasons, or because, in combination, one or more of the loci do not produce adequate product yield, or fragments which do not represent authentic alleles are produced in this reaction.
  • the following factors are preferably taken into consideration in deciding upon which loci to include in a multiplex of the present invention.
  • size ranges for alleles at each locus are determined. This information is used to facilitate separation of alleles between all the different loci, since any overlap could result in an allele from one locus being inappropriately identified as instability at another locus.
  • the amount of stutter exhibited by non-mononucleotide repeat loci is also preferably taken into consideration; as the amount of stutter exhibited by a locus can be a major factor in the ease and accuracy of interpretation of data. It is preferable to conduct a population study to determine the level of stutter present for each non-mononucleotide repeat locus. As noted above, tetranucleotide repeat markers display considerably less stutter that shorter repeat types like dinucleotides and therefore can be accurately scored in MSI assays (FIGS. 2 and 3)(Bacher & Schumm, 1998 Profiles in DNA 2(2):3-6).
  • At least one mononucleotide and at least two tetranucleotide repeat loci are included in the multiplex of MSI loci co-amplified according to the method or using the kit of the present invention, additional mononucleotide and/or tetranucleotide repeat loci can be included in the multiplex. It is also contemplated that multisatellite loci other than mono- or tetranucleotide repeat loci meeting the same or similar criteria to the criteria described above would be included in the multiplex.
  • the multiplex analyzed according to the present invention preferably includes a set of at least three MSI loci. It more preferably includes a set of at least five MSI loci, even more preferably a set of at least nine MSI loci.
  • the multiplex is a set of at least nine loci, it is preferably a set of at least the following loci: BAT-25, D10S1426, D3S2432, BAT-26, D7S3046, D7S3070, MONO-15, D1S518, and D7S1808, or more preferably a set of at least the following loci: BAT-25, D10S1426, D3S2432, BAT-26, D7S3046, D7S3070, BAT-40, MONO-15 and D7S1808.
  • a list of primers suitable for use in the first multiplex is provided in Table 11 of Example 6, below.
  • a list of primers suitable for use in the second, more preferred multiplex is provided in Table 13 of Example 8, below.
  • the genomic DNA amplified or co-amplified according to the methods of the present invention originates from biological material from an individual subject, preferably a mammal, more preferably from a dog, cat, horse, sheep, mouse, rat, rabbit, monkey, or human, even more preferably from a human or a mouse, and most preferably from a human being.
  • the biological material can be any tissue, cells, or biological fluid from the subject which contains genomic DNA.
  • the biological material is preferably selected from the group consisting of tumor tissue, disseminated cells, feces, blood cells, blood plasma, serum, lymph nodes, urine, and other bodily fluids.
  • the biological material can be in the form of tissue samples fixed in formalin and embedded in paraffin (hereinafter “PET”). Tissue samples from biopsies are commonly stored in PET for long term preservation. Formalin creates cross-linkages within the tissue sample which can be difficult to break, sometimes resulting in low DNA yields. Another problem associated with formalin-fixed paraffin-embedded samples is amplification of longer fragments is often problematic. When DNA from such samples is used in multiplex amplification reactions, a significant decrease in peak heights is seen with increasing fragment size.
  • the microsatellite analysis method and kit of the present invention are preferably designed to amplify and analyze DNA from PET tissue samples. (See Example 7 for an illustration of amplification of such samples using a method of the present invention.).
  • the method or kit of the present invention When the method or kit of the present invention is used in the analysis or detection of tumors, at least one sample of genomic DNA analyzed originates from a tumor.
  • a monomorphic or quasi-monomorphic locus such as MONO-11 or MONO-15 is amplified, the size of the resulting amplified alleles can be compared to the most commonly observed allele size at that locus in the general population.
  • the present method and kit is preferably used to diagnose or detect tumors by co-amplifying at least two different samples of DNA from the same individual, wherein one of the two samples originates from normal non-cancerous biological material.
  • microsatellite markers in DNA isolated from tumors were compared to microsatellite markers in DNA isolated from normal tissue or cells in order to detect MSI.
  • microsatellite loci were amplified from paired normal/tumor DNA samples and genotyped. If one or more different alleles were present in the tumor DNA sample that were not found in normal sample from the same individual, then it was scored as MSI positive. Dinucleotide, tetranucleotide and pentanucleotide repeat microsatellite markers were analyzed for frequency of alteration to determine the relative sensitivity of particular markers to MSI. Detailed information about the specific procedures used in this example are provided herein, below.
  • PCR and Microsatellite Analysis Fluorescently labeled primers from 275 microsatellite loci were used to amplify template DNA from normal/tumor pairs of samples. Two hundred and forty-five tetranucleotide repeat markers from the Research Genetics CHLC/Weber Human Screening Set Version 9.0 were evaluated (Research Genetics, Huntsville, Ala.).
  • Additional primer sets for tetranucleotide and pentanucleotide repeat markers were obtained from Promega Corporation (Madison, Wis.) (PowerPlexTM 16 System contains D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSFLPO, Penta D, vWA, D8S1179, TPOX, and FGA loci).
  • Microsatellite markers from the PowerPlexTM 16 System (Technical Manual #TMD012, Promega Corporation, Madison, Wis.) and dinucleotide repeat markers from the Microsatellite RER Assay system (see product literature from PE Biosystems, non Applied Biosystems, Foster City, Calif.) were analyzed following manufacturer's protocol.
  • PCR product Research Genetics markers were first diluted 1:4 in 1 ⁇ GoldST ⁇ R PCR buffer) was combined with 1 ⁇ l of Internal Lane Standard (Promega Corporation, Madison, Wis.) and 24 ⁇ l deionized formamide. Samples were denatured by heating at 95° C. for 3 minutes and immediately chilled on ice for 3 minutes. Separation and detection of amplified fragments was performed on an ABI PRISM®) 310 Genetic Analyzer following the standard protocol recommended in the User's Manual with the following settings: 5 second Injection Time, 15 kV Injection Voltage, 15 kV Run Voltage, 60° C. Run Temperature, and 28 minute Run Time.
  • Assay Interpretation Identification of normal and tumor allele sizes was accomplished by examining the appropriate electropherogram from the ABI PRISM 310 Genetic Analyzer (Applied Biosystems) and determining the predominant peaks for each locus. One or two peaks or alleles can be present for each locus in normal samples depending upon whether individual is homozygous or heterozygous for a particular marker. Allelic patterns or genotypes for normal and tumor pairs were compared and scored as MSI positive if one or more different alleles were present in the tumor DNA samples that were not found in normal sample from the same individual.
  • the tetra- and pentanucleotide repeat loci exhibited the smallest amount of stutter of the loci screened, above. See FIG. 4 for a plot of the % stutter results observed at the tetra- and pentanucleotide repeat loci.
  • the tetranucleotide repeat markers also varied in frequency of alteration, ranging from 0 to 100% MSI in the MSI-H group (FIG. 5). Pentanucleotide markers, in general, displayed low levels of MSI (FIG. 6).
  • Microsatellite markers showing high sensitivity to MSI >88% MSI with MSI-H samples) and high specificity ( ⁇ 8% MSI with MSI-L and MSS samples) with the CHTN samples were selected for further evaluation with 20 additional normal/tumor colon cancer samples from Mayo Clinic (Rochester, Minn.) (see Example 5).
  • flanking primers were designed for 33 GenBank DNA sequences using Oligo Primer Analysis Software version 6.0 (National Biosciences, Inc., Madison, Minn.) to amplify the region containing the poly (A) repeat. Evaluation of loci was performed using 9 MSI-H and 30 MSS colon cancer samples and corresponding normal DNA samples. Protocols for PCR, detection and analysis are described in Example 1.
  • loci Two characteristics were screened for in the new loci. First, loci were screened for which could detect MSI in the MSI-H group and not in the MSS group. Secondly, loci were selected on the basis of being monomorphic or nearly monomorphic (quasi-monomorphic). The monomorphic nature of the new loci was determined by genotyping 96 samples from 5 racial groups (African-American, Asian-American, Caucasian-American, Hispanic-American, Indian-American). Screening of 33 mononucleotide repeat loci revealed four new mononucleotide repeat loci (MONO-3, MONO-11, MONO-15, and MONO-19) that displayed high sensitivity to MSI (Table 4 and FIG.
  • a population study was conducted in which 93 samples from African-American individuals were genotyped using preferred microsatellite loci selected as candidates for multiplexing in the studies illustrated in Examples 1 and 2, above. See Table 6, below, and Table 3, above, for the amplification conditions used. See Table 7, below, for a list of the loci amplified and analyzed in this study.
  • a pooled Human Diversity DNA sample and control CEPH DNAs 1331-1 and 1331-2 were included in the screening population. African-American samples were used because they contain the greatest genetic diversity found in all racial groups.
  • primer-primer interactions that occurred when large number of oligos were combined in a single PCR reaction
  • primer design limitations due to sequence constraints at a particular locus (e.g., minimum size of amplicon allowed by DNA sequence, sub-optimal %GC of primers, difficulty balancing Tm's for all primers under uniform PCR conditions, difficulty in finding primers with desirable thermal profiles to minimize non-specific amplification, hairpin formation and self dimerization of primers, homology to other repeat sequences in human genome)
  • multiplex design allowing separation of all 9 loci within limited size range of 250 bp.
  • FIG. 9 A typical example of results produced from use of the nine locus MSI Multiplex described above is shown in FIG. 9.
  • the image was generated by simultaneous amplifying all nine selected microsatellite loci followed by separation of PCR products on an ABI 310 CE. Separation of all nine microsatellite loci in a single capillary (or gel lane) was accomplished by designing the multiplex so that loci would not overlap in size or through use of different fluorescent dyes.
  • the size ranges for the different multiplex loci were determined by genotyping 93 samples from African-American individuals using MSI Multiplex described following protocol described below.
  • a pooled Human Diversity DNA sample and control CEPH DNAs 1331-1 and 1331-2 were included in the screening population.
  • Protocol for MSI Multiplex Assay Template DNA from normal and tumor tissues obtained from same individual were purified using QIAamp Blood and Tissue Kit (QIAGEN, Santa Clarita, Calif.) following manufactures protocol. Two nanograms of template DNA in a 25 ⁇ l reaction volume was PCR amplified using protocol detailed in Table 12, below, using the cycling profile described in Table 3, above.
  • DNA was extracted from three 10 micron sections cut from PET blocks using QIAamp Tissue Kit (Qiagen, Santa Clarita, Calif.) according to the manufacture's instructions with the following modifications.
  • QIAamp Tissue Kit Qiagen, Santa Clarita, Calif.
  • Two microliters (out of 100 ⁇ l) of purified DNA solution was used as template for PCR reactions.
  • the nine locus multiplexed primer set described in Example 6 was used to amplify DNA from PET samples. The results indicate that the MSI Multiplex is capable of amplifying DNA from difficult and commonly used PET samples (FIG. 13).
  • a second set of nine loci was identified for analysis in a second MSI multiplex assay system, from eight of the best known set of loci identified in Example 4, above, for determining MSI in colon tumor samples, and from an additional mononucleotide repeat locus (BAT-40). Design of the multiplex loci was such that loci would not overlap in size within a single capillary or gel lane or could be labeled using different dyes when separated and overlap would occur. Using empirically determined optimal experimental conditions, multiplex microsatellite analysis was performed on several hundred healthy individuals.
  • Table 13 shows primer pairs used to amplify each locus in the second MSI multiplex, and approximate size range of fragments produced upon amplification of each locus with each primer pair.
  • the primer sequences identified in Table 13 were selected as being particularly suitable for amplification of each locus in the second MSI multiplex.
  • Results of MSI analysis on 36 MSI-H and 30 MSS colon cancer samples using the nine-locus multiplex and primer pairs described in Table 13 is shown in Table 14, below, and illustrated in FIG. 14.
  • FIG. 14 is a bar graph showing percent MSI measured at each locus of the multiplex from each of the two types of samples studied (i.e., MSI-H and MSS). An additional locus from the multiplex identified in Example 6, i.e. D1S518, was also included in this assay.
  • Protocol for MSI Multiplex Assay Two nanograms of normal template DNA were purified using QIAamp Blood and Tissue Kit (QIAGEN, Santa Clarita, Calif.) following the manufacturers protocol. DNA was amplified by PCR in a 25 ⁇ l reaction volume using protocol detailed in Table 15 and in Table 16, below. TABLE 15 Amplification Mix for MSI Multiplex Assay PCR Master Mix Component Volume Per Sample Nuclease Free Water 17.00 ⁇ l GoldST*R 10X Buffer (Promega) 2.50 ⁇ l Primer Pair Mix (10 ⁇ M) 2.50 ⁇ l AmpliTaq Gold DNA Polymerase (Perkin Elmer) 0.50 ⁇ l Template DNA (0.8 ng/ ⁇ l) 2.50 ⁇ l Total Reaction Volume 25.00 ⁇ l
  • Typical results of the paired tissue examples are shown in FIG. 16.
  • One or two alleles were present for each locus in normal samples depending upon whether individual was homozygous or heterozygous for a particular marker.
  • Allelic patterns or genotypes for normal and tumor pairs were compared and scored as MSI positive if one or more different alleles were present in the tumor DNA samples that were not found in normal sample from the same individual.

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US20100317534A1 (en) * 2009-06-12 2010-12-16 Board Of Regents, The University Of Texas System Global germ line and tumor microsatellite patterns are cancer biomarkers
US7902343B2 (en) 2000-09-15 2011-03-08 Promega Corporation Detection of microsatellite instability and its use in diagnosis of tumors
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US7749706B2 (en) 2010-07-06
WO2002086448A3 (fr) 2003-11-13
DE60235005D1 (de) 2010-02-25
EP1390538B1 (fr) 2010-01-06
EP1390538A4 (fr) 2005-08-03
AU2002303444A1 (en) 2002-11-05
US20070117136A1 (en) 2007-05-24
US20030180758A1 (en) 2003-09-25
EP1390538A2 (fr) 2004-02-25
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WO2002086448B1 (fr) 2003-12-11
WO2002086448A2 (fr) 2002-10-31

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