Detection of basepair substitution mutation at a frequency of 1 × 10−7 by combining two genotypic selection methods, MutEx enrichment and allele-specific competitive blocker PCR
✍ Scribed by Barbara L. Parsons; Robert H. Heflich
- Publisher
- John Wiley and Sons
- Year
- 1998
- Tongue
- English
- Weight
- 361 KB
- Volume
- 32
- Category
- Article
- ISSN
- 0893-6692
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✦ Synopsis
The detection of rare mutations has many important tant allele in a PCR reaction using a primer that has applications, including risk assessment of drugs and more mismatches to the wild-type allele than the chemicals, measuring environmental exposures to mutant allele. By combining these two approaches, genotoxins, and cancer cell detection. A sensitive the codon 61 mutation was detected at mutant genotypic selection method has been developed fractions as low as 1 in 10 7 . This sensitivity was that combines two different mutant allele selection achieved with the thermostable Thermus aquaticus techniques, MutEx enrichment and allele-specific MutS protein but not the Escherichia coli MutS procompetitive blocker PCR (ACB-PCR). This method tein. Using the combined approach, the average was developed and evaluated for the detection of Pfu DNA polymerase error rate { the standard error a CAA r AAA mutation at codon 61 of the mouse of the mean for this particular basepair was esti-H-ras gene. The MutEx enrichment is based on MutS mated to be 8 { 3 1 10 07 errors per duplication. binding to a mismatched basepair in heteroduplex The results indicate that MutEx/ACB-PCR is among DNA. The bound MutS protects the mutant allele the most sensitive genotypic selection methods for from degradation during subsequent exonuclease the detection of mutation. Environ.