Abstract
The presence of 5-methylcytosine as a modified base in DNA was discovered many decades ago. Surprisingly, however, and despite intense research efforts, the principal function of DNA methylation is still unknown. The CpG dinucleotide is the predominant if not exclusive target sequence for methylation by mammalian DNA methyltransferases. The analysis of DNA methylation at single-nucleotide resolution (genomic sequencing) has long been considered technically difficult, at least in mammalian cells. Recently, techniques have been developed that give a sufficient specificity and sensitivity for analysis of the methylation of single-copy genes by DNA-sequencing techniques (1,2). Currently, the most widely used method is based on bisulfite-induced deamination of cytosines followed by polymerase chain reaction (PCR) and DNA sequencing (2). Chemical DNA sequencing combined with ligation-mediated PCR (LM-PCR) is an alternative method for determination of genomic methylation patterns (1). LM-PCR is based on the ligation of an oligonucleotide linker onto the 5′ end of each DNA molecule that was created by a strand-cleavage reaction during chemical DNA sequencing. This ligation reaction provides a common sequence on all 5′ ends allowing exponential PCR to be used for signal amplification. One microgram of mammalian DNA per lane is more than sufficient to obtain good-quality DNA sequence ladders. The general LM-PCR procedure used for methylation analysis by chemical DNA sequencing is outlined in Fig. 1
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References
Pfeifer G. P., Steigerwald, S. D., Mueller, P. R., Wold, B., and Riggs, A. D. (1989) Genomic sequencing and methylation analysis by ligation mediated PCR. Science 246, 810–813.
Frommer, M., McDonald, M. E., Millar, D. S., Collis, C. M., Watt, F., Grigg, G. W., 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.
Pfeifer, G. P., Tanguay, R. L., Steigerwald, S. D., and Riggs, A. D. (1990) In vivo footprint and methylation analysis by PCR-aided genomic sequencing: comparison of active and inactive X chromosomal DNA at the CpG island and promoter of human PGK-1. Genes Dev. 4, 1277–1287.
Pfeifer, G. P., Steigerwald, S. D., Hansen, R. S., Gartler, S. M., and Riggs, A. D. (1990) Polymerase chain reaction-aided genomic sequencing of an X chromosome-linked CpG island: methylation patterns suggest clonal inheritance, CpG site autonomy, and an explanation of activity state stability. Proc. Natl. Acad. Sci. USA 87, 8252–8256.
Rideout III, W. M., Coetzee, G. A., Olumi, A. F., and Jones, P. A. (1990) 5-Methyl-cytosine as an endogenous mutagen in the human LDL receptor and p53 genes. Science 249, 1288–1290.
Reddy, P. M. S., Stamatoyannopoulos, G., Papayannopoulou, T., and Shen, C. K. J. (1994) Genomic footprinting and sequencing of human β-globin locus. J. Biol. Chem. 269, 8287–8295.
Hornstra, I. K. and Yang, T. P. (1994) High-resolution methylation analysis of the human hypoxanthine phosphoribosyltransferase gene 5′ region on the active and inactive X chromosomes: correlation with binding sites for transcription factors. Mol. Cell. Biol. 14, 1419–1430.
Tornaletti, S. and Pfeifer, G. P. (1995) Complete and tissue-independent methylation of CpG sites in the p53 gene: implications for mutations in human cancers. Oncogene 10, 1493–1499.
Szabó, P. E., Pfeifer, G. P., and Mann, J. R. (1998) Characterization of novel parental-specific epigenetic modifications upstream of the imprinted mouse H19 gene. Mol. Cell. Biol. 18, 6767–6776.
You, Y. H., Halangoda, A., Buettner, A., Hill, K., Sommer, S., and Pfeifer, G. P. (1998) Methylation of CpG dinucleotides in the lacI gene of the Big Blue’ transgenic mouse. Mutation Res. 420, 55–65.
Steigerwald, S. D., Pfeifer, G. P., and Riggs, A. D. (1990) Ligation-mediated PCR improves the sensitivity of methylation analysis by restriction enzymes and detection of specific DNA strand breaks. Nucleic Acids Res. 18, 1435–1439.
Rein, T., DePamphelis, M. L., and Zorbas, H. (1998) Identifying 5-methylcytosine and related modifications in DNA genomes. Nucleic Acids Res. 26, 2255–2264.
Warnecke, P. M., Stirzaker, C., Melki, J. R., Millar, D. S., Paul, C. L., and Clark, S. J. (1997) Detection and measurement of PCR bias in quantitative methylation analysis of bisulphite-treated DNA. Nucleic Acids Res. 25, 4422–4426
Stirzaker, C., Millar, D. S., Paul, C. L., Warnecke, P. M., Harrison, J., Vincent, P., et al. (1997) Extensive DNA methylation spanning the Rb promoter in retinoblas-toma tumors. Cancer Res. 57, 2229–2237.
Pfeifer, G. P. and Riggs, A. D. (1993) Genomic footprinting by ligation mediated polymerase chain reaction, In: Methods in Molecular Biology, vol. 15, (White, B.A., ed.), PCR Protocols: Current Methods and Applications, Humana Press, Totowa, NJ, pp. 153–168.
Tommasi, S. and Pfeifer, G. P. (1995) In vivo structure of the human cdc2 promoter: release of a p130/E2F-4 complex from sequences immediately upstream of the transcription initiation site coincides with induction of cdc2 expression. Mol. Cell. Biol. 15, 6901–6913.
Tornaletti, S. and Pfeifer, G. P. (1995) UV-light as a footprinting agent: modulation of UV-induced DNA damage by transcription factors bound at the promoters of three human genes. J. Mol. Biol. 249, 714–728.
Chin, P. L., Momand, J., and Pfeifer, G. P. (1997) In vivo evidence for binding of p53 to consensus binding sites in the p21 and GADD45 genes in response to ionizing radiation. Oncogene 15, 87–99.
Maxam, A. M. and Gilbert, W. (1980) Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 65, 499–560.
Denissenko, M. F., Chen, J. X., Tang, M. S., and Pfeifer, G. P. (1997) Cytosine methylation determines hot spots of DNA damage in the human P53 gene. Proc. Natl. Acad. Sci. USA 94, 3893–3898.
Törmänen, V. T. and Pfeifer, G. P. (1992) Mapping of UV photoproducts within ras protooncogenes in UV-irradiated cells: correlation with mutations in human skin cancer. Oncogene 7, 1729–1736.
Rychlik, W. and Rhoads, R. E. (1989) A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA. Nucleic Acids Res. 17, 8543–8551.
Komura, J.-I. and Riggs, A. D. (1998) Terminal transferase dependent PCR: a versatile and sensitive method for in vivo footprinting and detection of DNA adducts. Nucleic Acids Res. 26, 1807–1811.
Mueller, P. R. and Wold, B. (1989) In vivo footprinting of a muscle specific enhancer by ligation mediated PCR. Science 246, 780–786.
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Szabó, P.E., Mann, J.R., Pfeifer, G.P. (2002). Methylation Analysis by Chemical DNA Sequencing. In: Mills, K.I., Ramsahoye, B.H. (eds) DNA Methylation Protocols. Methods in Molecular Biology™, vol 200. Springer, Totowa, NJ. https://doi.org/10.1385/1-59259-182-5:029
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DOI: https://doi.org/10.1385/1-59259-182-5:029
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