Mutagenesis vol. 13 no. 6 pp. 581-588, 1998
© 1998 UK Environmental Mutagen Society/Oxford University Press
research-article |
Detection of a mouse H-ras codon 61 mutation using a modified allele-specific competitive blocker PCR genotypic selection method
Division of Genetic and Reproductive Toxicology, HFT-120, National Center for Toxicological Research 3900 NCTR Road, Jefferson, AR 72079, USA
A modified allele-specific competitive blocker PCR (ACB-PCR) has been developed as an approach for genotypic selection, the detection of a rare mutant allele based solely upon its altered nucleotide sequence. ACB-PCR genotypic selection operates through the preferential PCR amplification of mutant DNA using a primer that has more mismatches to the wild-type allele than the mutant allele. In addition, a blocker-primer with a 3'-terminal dideoxynucleotide and more mismatches to the mutant allele than the wild-type allele is incorporated to reduce the background and increase sensitivity. Using ACB-PCR, the CAA
AAA base substitution at codon 61 of the mouse H-ras gene was detected regularly at mutant fractions of 10–5. To accurately quantify the occurrence of this particular mutation, an internal amplification standard (AS) DNA was constructed. The H-ras and AS DNAs were subject to the same genotypic selection but were amplified using different upstream primers to give PCR products that can be distinguished by size. Defined mixtures of mutant and wild-type AS DNAs were used to study the effects of various components of the ACB-PCR. The concentration of dNTPs, blocker primer and Perfect Match® Polymerase Enhancer, as well as the choice of thermostable DNA polymerase and annealing temperature were examined. Conditions were identified for the concurrent detection of the CAAAAA mutation in the H-ras and AS DNAs. Using the identified conditions, approximately equal signals were obtained from equivalent amounts of the two DNA templates over a wide range of mutant fractions (1 in 10 to 1 in 105). This ACB-PCR method can be used for any application where it is necessary to quantify relatively small mutant fractions.
1To whom correspondence should be addressed. Tel: +1 870 543 7496; Fax: +1 870 543 7393; Email: bparsons{at}nctr.fda.gov
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