Abstract
Microsatellite instability has been shown to be relevant to various human diseases, including fragile X syndrome (1) and Huntington’s disease (2). In several human cancers, it has been reported that an increase or decrease in the number of repeat units between lymphocyte and tumor DNA derived from the same patient was found (3,4). Therefore, the analysis of microsatellite DNA has become necessary for the diagnosis of various diseases, especially hereditary and sporadic cancers (3,4). In hereditary nonpolyposis colorectal cancer (HNPCC), abnormalities of microsatellite DNA, (CA)n repeats, were reported to be an effective marker of microsatellite instability (5,6). The (CA)n repeats, which are related to HNPCC, are located on D2S123 marker DNA of the second chromosome. It is supposed that the alteration of (CA)n repeats might be associated with a defect in an early step of mismatch repair, and is controlled by mutator gene products such as hMSH2 (11). For the analysis of microsatellite instability, the PCR technique has been applied (5,8). DNA fragments containing (CA)n repeats can be amplified using specific primers (8; Fig. 1). The increase or decrease in the (CA) repeat number has been determined by evaluation of amplified DNA fragments by gel electrophoresis (8).
Detection of (CA)n repeat “D2S123” alterations. (A) Primers used for amplification of (CA)n repeats. The sizes of the amplified DNA were 180–350 bp. These specific sequences are given at the side of (CA)n repetitive sequence. (B) Amplified DNA fragments containing different (CA)n repeat numbers. An increase or decrease in the (CA)n repeat number brings about a change in the size of the PCR product directly.
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References
Pieretti, M., Zhang F., Fu, Y.-H., Warren, S. T., Oostra, B. A., Caskey, C. T., and Nelson, D. L. (1991) Absence of expression of the FMR-1 gene in fragile X syndrome. Cell 66, 817–822.
The Huntington’s disease collaborative research group. (1993) Novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosome. Cell 72, 971–983.
Han, H.-J., Yanagisawa, A., Kato, Y., Park, J.-G., and Nakamura, Y. (1994) Genetic instability in pancreatic cancer and poorly differentiated type of gastric cancer. Cancer Res. 53, 5087–5089.
Wooster, R., Cleton-Jansen, A. M., Collins, N., Mangion, J., Cornelis, R. S., Cooper, C. S., et al. (1994) Instability of short tandem repeats (microsatellites) in human cancer. Nature Genet. 6, 152–156.
Aaltonen, L. A., Peltomaki, P., Leach, F. S., Sistonen, P., Pylkkanen, L, Mecklin, J.-P., et al. (1993) Clues to the pathogenesis of famillial colorectal cancer. Science 260, 812–816.
Peltomaki, P., Lothe, R. A., Aaltonen, L. A., Pylkkanen, L., Nystrom-Lahti, M., Seruca, R., et al. (1993) Microsatellite instability is associated with tumors that characterize the hereditary non-polyposis colorectal carcinoma syndrome. Cancer Res. 53, 5853–5855.
Fishel, R., Lescoe, M. K., Rao, M. R. S., Copeland, N. G., Jenkins, N. A., Garber, J., Kane, M., and Kolodner, R. (1993) The human mutator gene homologue MSH2 and its association with hereditary nonpolyposis colon cancer. Cell 75, 1027–1038.
Wu, C., Akiyama, Y., Imai, K., Miyake, S., Nagasaki, H., Oto, M., et al. (1994) DNA alterations in cells from hereditary non-polyposis colorectal cancer patients. Oncogene 9, 991–994.
Martin, F., Vairelles, D., and Henrion, B. (1993) Automated ribosomal DNA fingerprinting by capillary electrophoresis of PCR products. Anal. Biochem. 63, 182–189.
Zhu, M., Hansen, D. L., Burd, S., and Gannon, F. (1989) Factors affecting freezone electrophoresis and isoelectric focusing in capillary electrophoresis. J. Chromatogr. 480, 311–319.
Oto, M., Suehiro, T., Akiyama, Y., and Yuasa, M. (1995) Microsatellite instability in cancer identified by non-gel sieving capillary electrophoresis. Clin. Chem. 41, 482–483.
Oto, M., Suehiro, T., and Yuasa, Y. (1995) Identification of mutated p53 in cancer by non-gel-sieving capillary electrophoretic SSCP analysis. Clin. Chem. 41, 1787–1788.
Oto, M., Suehiro, T., and Yuasa, Y. (1995) DNA hybridization analysis of PCR products by non-gel sieving capillary electrophoresis. PCR Methods. Applic. 4, 303–304.
Oto, M., Koguchi, K., and Yuasa, Y. (1997) Analysis of a polyadenine tract of the TGF-b type II receptor gene in colorectal cancers by non-gel sieving capillary electrophoresis. Clin. Chem. 43, 759–763.
Blin N. and Stafford, D. M. (1976) A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res. 3, 2303–2308.
Wittwer, C. T., Fillmore, G. C., and Hillyard, D. R. (1989) Automated polymerase chain reaction in capillary tubes with hot air. Nucleic Acids Res. 17, 4353–4357.
Wittwer, C. T., Fillmore, G. C., and Garling, D. J. (1990) Minimizing the time required for DNA amplification by efficient heat transfer to small samples. Anal. Biochem. 186, 328–331.
Talmadge, K. W., Zhu, M., Olech, L., and Siebert, C. (1996) Oligonucleotide analysis by capillary polymer sieving electrophoresis using acryloylamino-ethoxyethanol-coated capillaries. J. Chromatogr. 744, 347–354.
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Oto, M. (1999). Detection of Microsatellite Instability in Cancers by Means of Nongel-Sieving Capillary Electrophoresis. In: Palfrey, S.M. (eds) Clinical Applications of Capillary Electrophoresis. Methods in Molecular Medicine™, vol 27. Humana Press. https://doi.org/10.1385/1-59259-689-4:139
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DOI: https://doi.org/10.1385/1-59259-689-4:139
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