Article ID Journal Published Year Pages File Type
1176156 Analytical Biochemistry 2008 8 Pages PDF
Abstract

Polymerase-mediated single-base extension (SBE) of primers using a fluorescently labeled 2′,3′-dideoxynucleotide triphosphate terminator was originally commercialized as SNaPshot for analysis of single-nucleotide polymorphisms by capillary electrophoresis (CE). Application of this general method to bisulfite-converted/PCR-amplified genomic DNA (gDNA) to analytically infer polymorphic methylation status (i.e., 5-methylcytosine [5mC] vs. cytosine [C]) in CpG-rich regions of gDNA has been noted previously by others to be limited by CE mobility-shifted peaks for SBE products derived from guanine (G)/adenine (A)-mixed-base primers used to hybridize to possible polymorphic sites (i.e., C vs. thymine [T] resulting from 5mC vs. C, respectively). This limitation is precluded in the current study by using novel SNaPshot primers modified with N6-methoxy-2,6-diaminopurine (K), which was originally described in 1991 by Brown and Lin as a unique adenine–guanine analog capable of participating in three H-bonds with C or T in DNA. Oligonucleotides modified by K as a bispecific complement for C/T are commercially available or can be readily synthesized, and they may have utility in other assay formats used to analyze CpG methylation status.

Related Topics
Physical Sciences and Engineering Chemistry Analytical Chemistry
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