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Methylation Analysis by Chemical DNA Sequencing

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Part of the Methods in Molecular Biology™ book series (MIMB, volume 200)

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

Keywords

Sodium Acetate Methylation Pattern Bisulfite Sequencing Xylene Cyanol Sequence Ladder 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  1. 1.Department of BiologyBeckman Research Institute of the City of HopeDuarte

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