Bisulfite sequencing of cloned alleles.

Journal Article

Bisulfite sequencing of cloned alleles is a widely used method for capturing the methylation profiles of single alleles. This method combines PCR amplification of the bisulfite-modified DNA with the subcloning of the amplicons into plasmids followed by transformation into bacteria and plating on selective media. The resulting colony forming units are each comprised of bacterial clones containing the same plasmid reflecting a single allele in the original PCR reaction. Following whole cell PCR and sequencing, the results provide highly detailed information about the status of each CG site within an allele. Sequencing of a large number of individual clones can provide quantitative information, assuming unbiased PCR, subcloning and clone selection. The proportion of methylated cytosine at a particular position within the sequenced alleles can be determined by counting the number of alleles showing methylation at the position of interest and dividing this by the total number of clones sequenced.

Full Text

Duke Authors

Cited Authors

  • Huang, Z; Bassil, CF; Murphy, SK

Published Date

  • 2013

Published In

Volume / Issue

  • 1049 /

Start / End Page

  • 83 - 94

PubMed ID

  • 23913211

Electronic International Standard Serial Number (EISSN)

  • 1940-6029

Digital Object Identifier (DOI)

  • 10.1007/978-1-62703-547-7_8

Language

  • eng

Conference Location

  • United States