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Comment
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UnMethylCollector™ is performed on 200 ng of the Mse I digested human, male genomic DNA included in the kit. Enrichment of unmethylated CpG dinucleotides is confirmed using either endpoint or real time PCR analysis on the unbound and eluted fractions with the provided Xist and APC primers (Figures 1 & 2). The control DNA should have at least a 10-fold enrichment of unmethylated DNA bound and eluted from the protein complex as detected with the APC PCR primer mix. This means that of the total DNA recovered, there is ten times as much unmethylated DNA recovered in the eluted fraction as compared to the unbound fraction for the same locus. There should be less than 5% of unmethylated DNA detected from the eluted fraction with the Xist primer mix, as this is a methylated locus that should only be detected in the unbound fraction. APC, adenomatosis polyposis coli, is an unmethylated promoter in healthy tissues. Methylation of this promoter is associated with several types of cancers. The region amplified by this primer pair is 338 base pairs and contains 29 CpGs. Xist, X inactive specific transcript, is a methylated promoter in human, male genomic DNA, but is non-methylated in females. The region amplified by this primer pair is 178 base pairs and contains 8 CpGs.
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