Capillary electrophoretic analysis of methylation status in CpG-rich regions by single-base extension of primers modified with N6-methoxy-2,6-diaminopurine |
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Authors: | Boyd Victoria L Zon Gerald |
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Affiliation: | aApplied Biosystems, Foster City, CA 94404, USA |
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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. |
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Keywords: | Capillary electrophoresis Methylation status CpG-rich regions Bisulfite conversion Single-base extension K-base modified primers |
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