DNA Amplification

Bisulfite Sequencing

DNA methylation is an epigenetic mechanism known to play a role in mammalian gene regulation, genomic imprinting, and suppression of transposable elements. Methylation patterns can be characterized by interrogating specific loci or through genome-wide methylation profiling. Bisulfite treatment is considered the gold standard method for determining DNA methylation.

Sodium bisulfite deaminates unmodified cytosines to uracil but does not affect 5-methylcytosines. The bisulfite-treated DNA is then amplified by PCR, during which the 5-methylcytosines are amplified as cytosines and the uracils are amplified as thymines . DNA sequencing can then be used to elucidate the methylation status of a region of interest or an entire genome at single-nucleotide resolution.

PCR amplification of bisulfite-treated DNA requires an enzyme capable of tolerating uracil-containing DNA and high AT targets (conversion of unmodified cytosines to thymines increases the AT ratio of the target DNA). NEB’s EpiMark Taq DNA polymerase has been optimized for successful amplification of bisulfite-treated DNA.

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Protocols for Bisulfite Sequencing