Methylation-Dependent Fragment Separation: Novel Analysis Of 5-Methyl Cytosine By Capillary Electrophoresis Of Amplified Dna Using Pcr Incorporation Of Chemically Modified Dctp

Abstract

Methylation of the cytosine (C) ring to form 5-methyl cytosine (MeC) in normally unmethylated CpG-rich regions of promoters in genes is associated with transcriptional silencing. Quantification of MeC is of current interest in findining new biomarkers for cancer. To this end, and for basic research in epigenomics, we have investigated a new method for relatively simple measurement of MeC content by capillary electrophoresis (CE). PCR amplicons for CE analysis are generated from bisulfite-converted DNA [C --> uracil (U)] using fluorescently labeled primers that anneal independent of methylation status. Resultant incorporation of C vs. T at original MeC vs. C positions can lead to separate CE peaks for signal integration that is proportional to MeC content. Furthermore, these PCR products are suitable for additional methylation analyses by sequencing, single-base extension, or TaqMan. Interestingly, PCR using alpha -thio-dCTP led to greater CE separations.

Separation‐free single‐base extension assay with fluorescence resonance energy transfer for rapid and convenient determination of DNA methylation status at specific cytosine and guanine dinucleotide sites

“…DNA polymerase incorporates a single dideoxynucleotide triphosphate (ddNTP) that is complementary to the target base. Single base‐extended primer (i.e., SBE product) can be detected by various methods, including CE , MALDI‐TOF MS , surface enhanced Raman spectroscopy , and BeadChip array . However, all of these techniques are expensive, use high‐precision instruments, and require time‐consuming separation or cleanup/concentration of the SBE products.…”