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.
Applied Biosystems, Foster City, CA 94404, USA.
Victoria L
VL
Boyd
Gerald
G
Zon
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