Allele-specific competitive blocker PCR: a one-step method with applicability to pool screening.
We have developed a novel one-step pool screening PCR procedure which is based on the principles of amplification refractory mutation system (ARMS) and competitive oligonuleotide priming (COP) PCR. In addition to the usual primers, this approach uses two allele-specific competitive oligonucleotides, one of which is 3'-end labeled with a dideoxynucleotide and blocks amplification of the wild-type allele. An allele-specific product is generated only in the presence of the mutation. The introduction of an allele-specific competitive blocker oligonucleotide improves the specificity and robustness of ARMS-PCR. Further its sensitivity is dramatically increased, which allows detection of one mutant allele in a large excess of wild-type-bearing genomic DNA by electrophoresis in an ethidium bromide-stained agarose gel (up to 1 in 10(4) alleles). This makes the method ideal for nonradioactive pool screening. The successful application of the method has been demonstrated for four different point mutations, two in the apolipoprotein B gene (R3500Q, R3531C) which result in familial defective apolipoprotein B-100, one in the CFTR gene (R1162X), and one in the gene for lipoprotein lipase (G188E).
Orou
A
A
Institute for Medical Biology and Human Genetics, University of Innsbruck, Austria.
Fechner
B
B
Utermann
G
G
Menzel
H J
HJ
D000483:Alleles; D001483:Base Sequence; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D008071:Lipoprotein Lipase / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*
C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D008071:Lipoprotein Lipase