Degradable dUMP outer primers in merged tandem (M/T)-nested PCR: low- and single-copy DNA target amplification.
PCR amplification of DNA from a single initiating genomic molecule or low-copy template often requires two sequential amplification reactions with nested primer pairs to achieve the necessary specificity and sensitivity. Residual outer primers can result in undesired primer activity during the inner nested cycles. To circumvent this problem, we have used dU-containing primers for first round amplification and then uracil N-glycosylase (UNG) to degrade them and the ends of their dU-primer-containing amplified DNA products. We have applied this method to the detection of an exon 11 mutation in the HEXA gene. We have merged the step of a single-tube PCR amplification with outer dU primers with a tandem amplification using non-dU-nested primers (hence, the term merged tandem-nested or M/T-nested PCR). Serial dilutions of genomic DNA showed that this method could amplify a specific target from as few as three haploid genome equivalents of template DNA. Specific products were obtained from the DNA of single cells in 19 of 20 replicates, using 12 outer and 28 inner nested PCR cycles, with an intervening UNG digestion step. When coupled with heteroduplex mutational analysis, this method reliably distinguished mutant versus wild-type HEXA gene fragments amplified from single cells without primer artifact.
Grace
M B
MB
National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. marcy@codon.nih.gov
Buzard
G S
GS
Hughes
M R
MR
Gore-Langton
R E
RE
D016477:Artifacts; D004247:DNA / Q000032:analysis* / Q000235:genetics; D045647:DNA Glycosylases; D017931:DNA Primers / Q000737:chemistry* / Q000378:metabolism; D003856:Deoxyuracil Nucleotides; D018628:Gene Dosage; D020180:Heteroduplex Analysis; D006579:Heterozygote; D054818:Hexosaminidase A; D006801:Humans; D009699:N-Glycosyl Hydrolases / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D051981:Uracil-DNA Glycosidase; D001619:beta-N-Acetylhexosaminidases / Q000235:genetics*
D017931:DNA Primers; D003856:Deoxyuracil Nucleotides; D004247:DNA; C007267:2'-deoxyuridylic acid; D054818:Hexosaminidase A; D001619:beta-N-Acetylhexosaminidases; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase