Summary
Single-nucleotide polymorphism (SNP) genotyping can be carried out by annealing an oligonucleotide primer directly adjacent to the polymorphism and carrying out a single base extension using a polymerase reaction with labeled dideoxynucleotide triphosphates. This can be multiplexed by attaching a unique tag at the \(5\prime\)-end of each oligonucleotide primer and binding the corresponding antitag to a DNA microarray or microbead. After the polymerase reaction, the tag–antitag system can be used to demultiplex the experiment. However, such an assay requires careful primer and tag design to avoid any crossreactivity among the primers, tags, antitags, and template sequence. A procedure for designing the primers is described in this chapter.
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References
Cooper, D., Smith, B., Cooke, H., Niemann, S., and Schmidtke, J. (1985) An estimate of unique DNA sequence heterozygosity in the human genome. Hum. Genet. 69, 201–205.
Venter, J., Adams, M., Myers, E., Li, P., et al. (2001) The sequence of the human genome. Science 291, 1304–1351.
Syvänen, A.-C. (1999) From gels to chips: ‘minisequencing’ primer extension for analysis of point mutations and single nucleotide polymorphisms. Hum. Mutat. 13, 1–10.
Schafer, A. and Hawkins, J. (1998) DNA variation and the future of human genetics. Nat. Biotechnol. 16, 33–39.
Cai, H., White, P., Torney, D., Deshpande, A., Wang, Z., Marrone, B., and Nolan, J. (2000) Flow cytometry-based minisequencing: a new platform for high-throughput single-nucleotide polymorphism scoring. Genomics 66, 135–143.
SantaLucia, J., Jr. (1996). A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc. Natl. Acad. Sci. U. S. A. 95, 1460–1465.
Kaderali, L. and Schliep, A. (2002) Selecting signature oligonucleotides to identify organisms using DNA arrays. Bioinformatics 18, 1340–1349.
Kaderali, L., Deshpande, A., Nolan, J.P., and White, P.S. (2003) Primer-design for multiplexed genotyping. Nucleic Acids Res. 31, 1796–1802.
Zuker, M. (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415.
Waterman, M.S. (1995) Introduction to Computational Biology. Chapman 8 Hall, London.
Bommarito, S., Peyret, N., and SantaLucia, J., Jr. (2000) Thermodynamic parameters for DNA sequences with dangling ends. Nucleic Acids Res. 28, 1929–1934.
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© 2007 Humana Press
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Kaderali, L. (2007). Primer Design for Multiplexed Genotyping. In: Yuryev, A. (eds) PCR Primer Design. Methods in Molecular Biology™, vol 402. Humana Press. https://doi.org/10.1007/978-1-59745-528-2_13
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DOI: https://doi.org/10.1007/978-1-59745-528-2_13
Publisher Name: Humana Press
Print ISBN: 978-1-58829-725-9
Online ISBN: 978-1-59745-528-2
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