Capillary electrophoretic analysis of methylation status in CpG-rich regions by single-base extension of primers modified with N6-methoxy-2,6-diaminopurine.
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 N(6)-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.
Boyd
Victoria L
VL
Applied Biosystems, Foster City, CA 94404, USA.
Zon
Gerald
G
D015075:2-Aminopurine / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D018899:CpG Islands / Q000235:genetics*; D019175:DNA Methylation; D017931:DNA Primers / Q000235:genetics* / Q000378:metabolism*; D019075:Electrophoresis, Capillary; D015894:Genome, Human / Q000235:genetics; D006801:Humans; D020641:Polymorphism, Single Nucleotide
D017931:DNA Primers; C532014:N6-methoxy-2,6-diaminopurine; D015075:2-Aminopurine