Abstract
Pyrosequencing® technique has been widely used to perform both single-nucleotide polymorphism detection and quantitative DNA methylation analysis. Simplex Pyrosequencing is sufficient to interrogate more than one polymorphic site if these gene variants are within the reach of the sequencing reaction. For polymorphisms far apart from each other or located on different genes, multiple simplex analyses are required. To reduce the number of simplex reactions, multiplex Pyrosequencing becomes a useful alternative method. The multiplex reaction is performed in the presence of single or multiple templates with several sequencing primers. Factors such as primer selection for the PCR and Pyrosequencing reaction, generation of optimal nucleotide dispensation order, use of internal and external controls, preparation of instrumentation, and Pyrogram® interpretation are essential to the success of the multiplexing. In this chapter, the mouse 45S rRNA gene is used to present two general multiplex Pyrosequencing protocols for determining DNA methylation and allele frequency in the spacer promoter region of this gene.
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References
O’Meara, D., Wilbe, K., Leitner, T., Hejdeman, B., Albert, J., and Lundeberg, J. (2001) Monitoring resistance to human immunodeficiency virus type 1 protease inhibitors by Pyrosequencing. J. Clin. Microbiol. 39, 464–473.
Ronaghi, M. and Elahi, E. (2002) Pyrosequencing for microbial typing. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 25, 67–72.
Ferraris, A., Rappaport, E., Santacroce, R., et al. (2002) Pyrosequencing for detection of mutations in the Connexin 26 (GJB2) and Mitochondrial 12S RNA (MTRNR1) genes associated with hereditary hearing loss. Human Mutat. 20, 312–320.
Berg, L. M., Sanders, R., and Alderborn, A. (2002) Pyrosequencing technology and the need for versatile solutions in molecular clinical research. Expert Rev. Mol. Diagn. 2, 361–369.
Colella, S., Shen, L., Baggerly, K. A., Issa, J. P., and Krahe, R. (2003) Sensitive and quantitative universal Pyrosequencing methylation analysis of CpG sites. Biotechniques 35, 146–150.
Zackrisson, A. and Lindblom, B. (2003) Identification of CYP2D6 alleles by single nucleotide polymorphism analysis using Pyrosequencing. Eur. J Clin Pharmacol. 59, 521–526.
Ahluwalia, R., Freimuth, R., McLeod, H. L., and Marsh S. (2003) Use of Pyrosequencing to detect clinically relevant polymorphisms in dihydropyrimidine dehydrogenase. Clin. Chem. 49, 1661–1664.
Ronaghi, M., Uhlen, M., and Nyren, P. (1998) A sequencing method based on real-time pyrophosphate. Science 281, 363–365.
Alderborn, A., Kristofferson, A., and Hammerling, U. (2000) Determination of single-nucleotide polymorphisms by real-time pyrophosphate DNA sequencing. Genome Res. 10, 1249–1258.
Ahmadian, A., Gharizadeh, B., Gustafsson, A.-C., et al. (2000) Single-nucleotide polymorphism analysis by Pyrosequencing. Anal. Biochem. 280, 103–110.
Fakhrai-Rad, H., Pourmand, N., and Ronaghi, M. (2002) Pyrosequencing™: An accurate detection platform for single nucleotide polymorphisms. Human Mutat. 19, 479–485.
Ronaghi, M. (2003) Pyrosequencing for SNP genotyping. In: Single Nucleotide Polymorphisms, (Kwok, P.-Y., ed.), Humana Press, Totowa, NJ, pp. 189–195.
Langaee, T. and Ronaghi, M. (2005) Genetic variation analyses by Pyrosequencing. Mutat Res. 573, 96–102.
Dupont J. M., Tost, J., Jammes, H., and Gut, I. G. (2004) De novo quantitative bisulfite sequencing using the Pyrosequencing technology. Anal. Biochem. 333, 119–127.
Shiao, Y.-H., Crawford, E. B., Anderson, L. M., Patel, P., and Ko, K. (2005) Allele specific germ cell epimutation in the spacer promoter of the 45S ribosomal RNA gene after Cr(III) exposure. Toxicol. Appl. Pharmacol. 205, 290–296.
Pourmand, N., Elahi, E., Davis, R. W., and Ronaghi, M. (2002) Multiplex Pyrosequencing. Nucleic Acids Res. 30, e31.
Lötsch, J., Skarke, C., and Geisslinger, G. (2003) Simultaneous screening for the three mutations in the ABCB1 gene. Genomics 82, 503–510
Palmieri, O., Toth, S., Ferraris, A., et al. (2003) Card15 genotyping in inflammatory bowel disease patients by multiplex Pyrosequencing. Clin. Chem. 49, 1675–1679.
Pyrosequencing Technical note 105. URL: http://www.Pyrosequencing.com.
Dunker, J., Larsson, U., Petersson, D., et al. (2003) Parallel DNA template preparation using a vacuum filtration sample transfer device. Biotechniques 34, 862–868.
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Patel, P., Shiao, YH., Fortina, P. (2007). Multiplex Pyrosequencing® for DNA Variation Analysis. In: Walker, J.M., Marsh, S. (eds) Pyrosequencing® Protocols. Methods in Molecular Biology™, vol 373. Humana Press. https://doi.org/10.1385/1-59745-377-3:75
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DOI: https://doi.org/10.1385/1-59745-377-3:75
Publisher Name: Humana Press
Print ISBN: 978-1-58829-645-0
Online ISBN: 978-1-59745-377-6
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