Abstract
Most available protocols for gene-specific DNA methylation analysis are either labor intensive, not quantitative, or limited to the measurement of the methylation status of only one or very few CpG positions. Pyrosequencing is a real-time sequencing technology that overcomes these limitations. After bisulfite modification of genomic DNA, a region of interest is amplified by polymerase chain reaction (PCR) with one of the two primers being biotinylated. The PCR-generated template is rendered single stranded and a pyrosequencing primer is annealed to analyze quantitatively CpGs within 120 bases. Advantages of the pyrosequencing technology are the ease of its implementation, the high quality and the quantitative nature of the results, and its ability to identify differentially methylated positions in close proximity. A minimum amount of 10 ng of bisulfite-treated DNA is necessary to obtain high reproducibility and avoid random amplification. The required DNA amount can be provided by an individual sample or a pool of samples to rapidly investigate the presence of variable DNA methylation patterns. The use of pools and serial pyrosequencing, that is, the successive use of several pyrosequencing primers on the same DNA template, significantly reduces cost, labor, and analysis time as well as saving precious DNA samples for the analysis of gene-specific DNA methylation patterns.
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References
Ronaghi, M. (2001) Pyrosequencing sheds light on DNA sequencing. Genome Res 11, 3–11.
Ronaghi, M., Uhlen, M., Nyren, P. (1998) A sequencing method based on real-time pyrophosphate. Science 281, 363–365.
Ahmadian, A., Ehn, M., Hober, S. (2006) Pyrosequencing: history, biochemistry and future. Clin Chim Acta 363, 83–94.
Sanger, F., Nicklen, S., Coulson, A. R. (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74, 5463–5467.
Marsh, S. (2007) Pyrosequencing Protocols. Methods in Molecular Biology 373, Humana Press, Totowa, NJ.
Wasson, J. (2007) Allele quantification and DNA pooling methods. Methods Mol Biol 373, 63–74.
Pielberg, G., Day, A. E., Plastow, G. S., et al. (2003) A sensitive method for detecting variation in copy numbers of duplicated genes. Genome Res 13, 2171–2177.
Deutsch, S., Choudhury, U., Merla, G., et al. (2004) Detection of aneuploidies by paralogous sequence quantification. J Med Genet 41, 908–915.
Colella, S., Shen, L., Baggerly, K. A., et al. (2003) Sensitive and quantitative universal Pyrosequencing methylation analysis of CpG sites. Biotechniques 35, 146–150.
Tost, J., Dunker, J., Gut, I. G. (2003) Analysis and quantification of multiple methylation variable positions in CpG islands by Pyrosequencing. Biotechniques 35, 152–156.
Uhlmann, K., Brinckmann, A., Toliat, M. R., et al. (2002) Evaluation of a potential epigenetic biomarker by quantitative methyl-single nucleotide polymorphism analysis. Electrophoresis 23, 4072–4079.
Frommer, M., McDonald, L. E., Millar, D. S., et al. (1992) A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci USA 89, 1827–1831.
Dupont, J. M., Tost, J., Jammes, H., et al. (2004) De novo quantitative bisulfite sequencing using the pyrosequencing technology. Anal Biochem 333, 119–127.
Chelbi, S. T., Mondon, F., Jammes, H., et al. (2007) Expressional and epigenetic alterations of placental serine protease inhibitors: SERPINA3 is a potential marker of preeclampsia. Hypertension 49, 76–83.
Dejeux, E., Audard, V., Cavard, C., et al. (2007) Rapid identification of promoter hypermethylation in hepatocellular carcinoma by pyrosequencing of etiologically homogeneous sample pools. J Mol Diagn 9, 510–520.
Issa, J. P., Gharibyan, V., Cortes, J., et al. (2005) Phase II study of low-dose decitabine in patients with chronic myelogenous leukemia resistant to imatinib mesylate. J Clin Oncol 23, 3948–3956.
Yang, A. S., Estecio, M. R., Doshi, K., et al. (2004) A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements. Nucleic Acids Res 32, e38.
Mirmohammadsadegh, A., Marini, A., Nambiar, S., et al. (2006) Epigenetic silencing of the PTEN gene in melanoma. Cancer Res 66, 6546–6552.
Xinarianos, G., McRonald, F. E., Risk, J. M., et al. (2006) Frequent genetic and epigenetic abnormalities contribute to the deregulation of cytoglobin in non-small cell lung cancer. Hum Mol Genet 15, 2038–2044.
Kwabi-Addo, B., Chung, W., Shen, L., et al. (2007) Age-related DNA methylation changes in normal human prostate tissues. Clin Cancer Res 13, 3796–3802.
Rodriguez-Canales, J., Hanson, J., Tangrea, M., et al. (2007) Identification of a unique epigenetic sub-microenvironment in prostate cancer. J Pathol 211, 410–419.
White, H. E., Durston, V. J., Harvey, J. F., et al. (2006) Quantitative analysis of SNRPN(correction of SRNPN) gene methylation by pyrosequencing as a diagnostic test for Prader-Willi syndrome and Angelman syndrome. Clin Chem 52, 1005–1013.
Wong, H. L., Byun, H. M., Kwan, J. M., et al. (2006) Rapid and quantitative method of allele-specific DNA methylation analysis. Biotechniques 41, 734–739.
Tost, J., Gut, I. G. (2007) Analysis of gene-specific DNA methylation patterns by pyrosequencing technology. Methods Mol Biol 373, 89–102.
Tost, J., El abdalaoui, H., Gut, I.G. (2006) Serial pyrosequencing for quantitative DNA methylation analysis. Biotechniques 40, 721–726.
Li, L. C., Dahiya, R. (2002) MethPrimer: designing primers for methylation PCRs. Bioinformatics 18, 1427–1431.
Dunker, J., Larsson, U., Petersson, D., et al. (2003) Parallel DNA template preparation using a vacuum filtration sample transfer device. Biotechniques 34, 862–868.
Aranyi, T., Varadi, A., Simon, I., et al. (2006) The BiSearch web server. BMC Bioinformatics 7, 431.
Warnecke, P. M., Stirzaker, C., Melki, J. R., et al. (1997) Detection and measurement of PCR bias in quantitative methylation analysis of bisulphite-treated DNA. Nucleic Acids Res 25, 4422–4426.
Tetzner, R., Dietrich, D., Distler, J. (2007) Control of carry-over contamination for PCR-based DNA methylation quantification using bisulfite treated DNA. Nucleic Acids Res 35, e4.
Acknowledgments
This work was supported by the French Ministry of Research and the European Commission under the Integrated Project “MolPage” (contract number LSHG-CT-2004-512966).
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Dejeux, E., abdalaoui, H.E., Gut, I.G., Tost, J. (2009). Identification and Quantification of Differentially Methylated Loci by the Pyrosequencing™ Technology. In: Tost, J. (eds) DNA Methylation. Methods in Molecular Biology, vol 507. Humana Press. https://doi.org/10.1007/978-1-59745-522-0_15
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DOI: https://doi.org/10.1007/978-1-59745-522-0_15
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