![]() The sequence data is semi-quantitative and can reveal variable patterns of methylation within a specimen. Relative to direct sequencing of a PCR amplicon pool generated from a bisulfite-modified genomic DNA template, sequencing of clones provides a higher resolution result due to the ability to score methylation for single alleles. Accuracy increases with the number of clones sequenced. Methylation percentages for each of the CG sites are often calculated according to the number of methylated and unmethylated CG cytosines at a given position across the different clonesĪnastasia Malek and Oleg Tchernitsa (eds.), Ovarian Cancer: Methods and Protocols, Methods in Molecular Biology, vol. A portion of the PCR product is subcloned into plasmids and randomly selected clones are sequenced to provide the methylation status of the CG sites within each of the individual DNA molecules. The amplified PCR product comprises a pool of DNA molecules representing the individual alleles present within the DNA specimen used for PCR. ![]() The DNA sequence under investigation is amplified by PCR with primers specific for one strand of the bisulfite converted DNA. The genomic DNA is first modified by sodium bisulfite. Introduction The cloning and sequencing of bisulfite converted DNA is one of the gold standards of DNA methylation analysis, as the sequencing of subcloned individual DNA molecules provides detailed information on the methylation status of every CG site within a given allele for relatively long stretches of DNA sequence. Key words DNA methylation, Sodium bisulfite, Cloning, Capillary sequencing 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. Sequencing of a large number of individual clones can provide quantitative information, assuming unbiased PCR, subcloning and clone selection. Following whole cell PCR and sequencing, the results provide highly detailed information about the status of each CG site within an allele. The resulting colony forming units are each comprised of bacterial clones containing the same plasmid reflecting a single allele in the original PCR reaction. 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. Murphy Abstract Bisulfite sequencing of cloned alleles is a widely used method for capturing the methylation profiles of single alleles. Chapter 8 Bisulfite Sequencing of Cloned Alleles Zhiqing Huang, Christopher F. ![]()
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