Methylation-dependent fragment separation: novel analysis of 5-methyl cytosine by capillary electrophoresis of amplified DNA using PCR incorporation of chemically modified dCTP.
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.
Boyd
Victoria L
VL
Genetic Analysis R&D, Foster City, California 94404, USA.
Moody
Kristina I
KI
Karger
Achim E
AE
Livak
Kenneth J
KJ
Zon
Gerald
G
Burns
John W
JW
D044503:5-Methylcytosine / Q000032:analysis*; D018899:CpG Islands; D004247:DNA / Q000032:analysis / Q000302:isolation & purification*; D003845:Deoxycytosine Nucleotides / Q000737:chemistry*; D019075:Electrophoresis, Capillary; D006801:Humans; D008745:Methylation; D016133:Polymerase Chain Reaction / Q000379:methods*
D003845:Deoxycytosine Nucleotides; C024107:2'-deoxycytidine 5'-triphosphate; D044503:5-Methylcytosine; D004247:DNA