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Homogeneous Allele-Specific PCR in SNP Genotyping

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Single Nucleotide Polymorphisms

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 212))

Abstract

Allele-specific (AS) PCR amplification (1–3) has been used in combination with gel based detection to genotype-specific polymorphisms. Until recently a major drawback of this method was that it was labor-intensive and without high-throughput instrumentation (4). The single nucleotide polymorphism (SNP) genotyping assay presented here combines AS PCR amplification with kinetic, realtime monitoring (5–6). It is robust, rapid, inexpensive, and allows accurate measurement of allele frequencies in pools of DNA, facilitating large-scale gene mapping.

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References

  1. Newton, C. R., Graham, A., Heptinstall, L. E., Powell, S. J., Summers, C., Kalsheker, N., et al. (1989) Analysis of any point mutation in DNA: the amplification refractory mutation system (ARMS). Nucleic Acids Res. 17, 2503–2516.

    Article  PubMed  CAS  Google Scholar 

  2. Sommer, S. S., Cassady, J. D., Sobell, J. L., and Bottema, C. D. (1989) A novel method for detecting point mutations or polymorphisms and its application to population screening for carriers of phenylketonuria. Mayo Clin. Proc. 64, 1361–1372.

    PubMed  CAS  Google Scholar 

  3. Wu, D. Y., Ugozolli, L., Pal, B. K., and Wallace, R. B. (1989). Proc.Allele-specific enzymatic amplification of beta-globin genomic DNA for diagnosis of sickle-cell anemia. Proc. Natl. Acad. Sci. USA 86, 2757–2760.

    Article  PubMed  CAS  Google Scholar 

  4. Landegren, U., Nilsson, M., and Kwok, P.-Y. (1998) Reading bits of genetic information: methods for single-nucleotide polymorphism analysis. Genome Res. 8, 769–776.

    PubMed  CAS  Google Scholar 

  5. Germer, S. and Higuchi, R. (1999) Single-tube genotyping without oligonucleotide probesGenome. Genome Res. 9, 72–78.

    PubMed  CAS  Google Scholar 

  6. Germer, S., Holland, M. J., and Higuchi, R. (2000) High-throughput SNP allele-frequency determination in pooled DNA samples by kinetic PCR. Genome Res. 10, 258–266.

    Article  PubMed  CAS  Google Scholar 

  7. Chou, Q., Russel, M., Birch, D. E., Raymond, J., and Block, W. (1992) Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplification. Nucleic Acids Res. 20, 1717–1723.

    Article  PubMed  CAS  Google Scholar 

  8. Lawyer, F. C., Stoffel, S., Saiki, R. K., 0Chang, S. Y., Landre, P. A., Abramson, R. D., and Gelfand, D. H. (1993) High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5′ to 3′ exonuclease. PCR Methods Appl. 2, 275–287.

    PubMed  CAS  Google Scholar 

  9. Tada, M., Omata, M., Kawai, S., Saisho, H., Ohto, M., Saiki, R. K., and Sninsky, J. J. (1993) Detection of ras gene mutations in pancre-atic juice and peripheral blood of patients with pancreatic adenocar-cinoma. Cancer Res. 53, 2472–2474.

    PubMed  CAS  Google Scholar 

  10. Birch, D. E. (1996) Simplified hot start PCR. Nature 381, 445–446.

    Article  PubMed  CAS  Google Scholar 

  11. Grupe, A., Germer, S., Usuka, J., Aud, D., Belknap, J. K., Klein, R. F., et al. (2001) In-silico mapping of complex disease-related traits in mice. Science 292, 1915–1918.

    Article  PubMed  CAS  Google Scholar 

  12. Wang, D. G., Fan, J.-B., Siao, C.-J., Berno, A., Young, P., Sapolsky, R., et al. (1998) Large-scale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome. Science 280, 1077–1082.

    Article  PubMed  CAS  Google Scholar 

  13. Arnheim, N., Strange, C., and Erlich, H. (1985) Use of pooled DNA samples to detect linkage disequilibrium of polymorphic restriction fragments and human disease: studies of the HLA class II loci. Proc. Natl. Acad. Sci. USA 82, 6970–6974.

    Article  PubMed  CAS  Google Scholar 

  14. Barcellos, L. F., Klitz, W., Field, L. L., Tobias, R., Bowcock, A. M., Wilson, R., et al. (1997) Association mapping of disease loci, by use of pooled DNA genomic screen. Am. J. Hum. Genet. 61, 734–747.

    Article  PubMed  CAS  Google Scholar 

  15. Breen, G., Harold, D., Ralston, S., Shaw, D., and St. Clair, D. (2000) Determining SNP allele frequencies in DNA pools. Biotechniques 28(3), 464–466.

    PubMed  CAS  Google Scholar 

  16. Kwok, P.-Y., Carlson, C., Yager, T. D., Ankener, W., and Nickerson, D. A. (1994) Comparative analysis of human DNA variations by fluo-rescence-based sequencing of PCR products. Genomics 23, 138–144.

    Article  PubMed  CAS  Google Scholar 

  17. Kwok, P.-Y. (2000) Approaches to allele frequency determination. Pharmacogenomics 1(2), 231–235.

    Article  PubMed  CAS  Google Scholar 

  18. Pacek, P., Sajantila, A., and Syvänen, A.-C. (1993) Determination of allele frequencies at loci with length polymorphism by quantitative analysis of DNA amplified from pooled samples. PCR Methods Applic. 2, 313–317.

    CAS  Google Scholar 

  19. Sasaki, T., Tahira, T., Suzuki, A., Higasa, K., Kukita, Y., Baba, S., and Hayashi, K. (2001) Precise estimation of allele frequencies of single-nucleotide polymorphisms by a quantitative SSCP analysis of pooled DNA. Am. J. Hum. Genet. 68, 214–218.

    Article  PubMed  CAS  Google Scholar 

  20. Shaw, S. H., Carrasquillo, M. M., Kashuk, C., Puffenberger, E. G., and Chakravarti, A. (1998) Allele frequency distributions in pooled DNA samples: applications to mapping complex disease genes. Genome Res. 8, 111–123.

    PubMed  CAS  Google Scholar 

  21. Chen, J., Higuchi, R., Germer, S., Berkowitz, G., Godbold, J., and Wetmur, J. G. (2001) Kinetic PCR on pooled DNA: a high-through-put, high-efficiency alternative in genetic epidemiologic studies. Cancer Epidemiol. Biomark. Prevent. 11, 131–136.

    Google Scholar 

  22. Ririe, K. M., Rasmussen, R. P., and Wittwer, C. T. (1997) Product differentiation by analysis of DNA melting curves during the poly-merase chain reaction. Anal. Biochem. 245, 154–160.

    Article  PubMed  CAS  Google Scholar 

  23. Bernard, P. S., Lay, M. J., and Wittwer, C. T. (1998) Integrated ampli-fication and detection of the C677T point mutation in the methylene-tetrahydrofolate reductase gene by fluorescence resonance energy transfer and probe melting curves. Anal. Biochem. 255, 101–107.

    Article  PubMed  CAS  Google Scholar 

  24. Chen, X., Livak, K. J., and Kwok, P.-Y. (1998) A homogeneous, ligase-mediated DNA diagnostic test. Genome Res. 8, 549–556.

    PubMed  CAS  Google Scholar 

  25. Fiandaca, M. J., Hyldig-Nielsen, J. J., Gildea, B. D., and Coull, J. M. (2001) Self-reporting PNA/DNA primers for PCR analysis. Genome Res. 11, 609–613.

    Article  PubMed  CAS  Google Scholar 

  26. Holland, P. M., Abramson, R. D., Watson, R., and Gelfand, D. H. (1991) Detection of specific polymerase chain reaction product by utilizing the ′’?3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. USA 88, 7276–7280.

    Article  PubMed  CAS  Google Scholar 

  27. Kostrikis, L. G., Tyagi, S., Mhlanga, M. M., Ho, D. D., and Kramer, F. R. (1998) Spectral genotyping of human alleles. Science 279, 1228–1229.

    Article  PubMed  CAS  Google Scholar 

  28. Tyagi, S. and Kramer, F. R. (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14, 303–308.

    Article  PubMed  CAS  Google Scholar 

  29. Whitcombe, D., Theaker, J., Guy, S. P., Brown, T., and Little, S. (1999) Detection of PCR products using self-probing amplicons and fluorescence. Nat. Biotechnol. 17, 804–807.

    Article  PubMed  CAS  Google Scholar 

  30. Beaudet, L., Bedard, J., Breton, B., Mercuri, R. J., and Budarf, M. L. (2001) Homogenous assays for single-nucelotide polymorphism typ-ing using AlphaScreen. Genome Res. 11, 600–608.

    Article  PubMed  CAS  Google Scholar 

  31. Jeffreys, A. J., MacLeod, A., Tamaki, K., Neil, D. L., and Monckton, D. G. (1991) Minisatellite repeat coding as a digital approach to DNA typing. Nature 354, 204–209.

    Article  PubMed  CAS  Google Scholar 

  32. Myakishev, M. V., Khripin, Y., Hu, S., and Hamer, D. H. (2001) High-throughput SNP genotyping by allele-specific PCR with uni-versal energy-transfer-labelled primers. Genome Res. 11, 163–169.

    Article  PubMed  CAS  Google Scholar 

  33. Neilan, B. A., Wilton, A. N., and Jacobs, D. (1997) A universal pro-cedure for primer labelling of amplicons. Nucleic Acids Res. 25, 2938–2939.

    Article  PubMed  CAS  Google Scholar 

  34. Whitcombe, D., Brownie, J., Gillard, H. L., McKechnie, D., Theaker, J., Newton, C. R., and Little, S. (1998) A homogeneous fluorescence assay for PCR amplicons: its application to real-time, single-tube genotyping. Clin. Chem. 44, 918–923.

    PubMed  CAS  Google Scholar 

  35. Winn-Deen, E. S. (1998) Direct fluorescence detection of allele-spe-cific PCR products using novel energy-transfer labeled primers. Mol. Diagn. 3, 217–221.

    Article  PubMed  CAS  Google Scholar 

  36. Higuchi, R. (1989) Simple and rapid preparation of samples for PCR, in PCR Technology: Principles and Applications for DNA Amplification (Ehrlich, H. A., ed.), M. Stockton Press, New York, NY, pp. 31–38.

    Google Scholar 

  37. Helmuth, R., Fildes, N., Blake, E., Luce, M. C., Chimera, J., Madej, R., et al. (1990) HLA-DQα allele and genotype frequencies in vari-ous human populations, determined by using enzymatic amplifica-tion and oligonucleotide probes. Am. J. Hum. Genet. 47, 515–523.

    PubMed  CAS  Google Scholar 

  38. Miller, S. A., Dykes, D. D., and Poleskly, H. F. (1988) A simple salt-ing out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16(3), 1215.

    Article  PubMed  CAS  Google Scholar 

  39. Wetmur, J. G. (1991) DNA probes: applications of the principles of nucleic acid hybridization. Crit. Rev. Biochem. Mol. Biol. 26, 227–259.

    Article  PubMed  CAS  Google Scholar 

  40. Breslauer, K. J., Frank, R., Blocker, H., and Marky, L. A. (1986) Predicting DNA duplex stability from the base sequence. Proc. Natl. Acad. Sci. USA 83(11), 3746–3750.

    Article  PubMed  CAS  Google Scholar 

  41. Beasley, E. M., Myers, R. M., Cox, D. R., and Lazzeroni, L. C. (1999) Statistical refinement of primer design parameters, in PCR Applica-tions: Protocols for Functional Genomics (Innis, M. A., Gelfand, D. H., and Sninsky, J. J., eds.), Academic Press, San Diego, CA.

    Google Scholar 

  42. Longo, M. C., Berninger, M. S., and Hartley, L. L. (1990) Use of uracil DNA glycosylase to control carry-over contamination in poly-merase chain reactions. Gene 93(1), 125–128.

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Roche Molecular Systems, Alameda, CA

    Søren Germer & Russell Higuchi

Authors
  1. Søren Germer
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  2. Russell Higuchi
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Editor information

Editors and Affiliations

  1. Cardiovascular Research Institute and Department of Dermatology, University of California, San Francisco, San Francisco, CA, USA

    Pui-Yan Kwok MD, PhD

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© 2003 Humana Press Inc.

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Germer, S., Higuchi, R. (2003). Homogeneous Allele-Specific PCR in SNP Genotyping. In: Kwok, PY. (eds) Single Nucleotide Polymorphisms. Methods in Molecular Biology™, vol 212. Springer, Totowa, NJ. https://doi.org/10.1385/1-59259-327-5:197

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  • DOI: https://doi.org/10.1385/1-59259-327-5:197

  • Publisher Name: Springer, Totowa, NJ

  • Print ISBN: 978-0-89603-968-1

  • Online ISBN: 978-1-59259-327-9

  • eBook Packages: Springer Protocols

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