Detection of basepair substitution mutation at a frequency of 1 x 10(-7) by combining two genotypic selection methods, MutEx enrichment and allele-specific competitive blocker PCR.
The detection of rare mutations has many important applications, including risk assessment of drugs and chemicals, measuring environmental exposures to genotoxins, and cancer cell detection. A sensitive genotypic selection method has been developed that combines two different mutant allele selection techniques, MutEx enrichment and allele-specific competitive blocker PCR (ACB-PCR). This method was developed and evaluated for the detection of a CAA --> AAA mutation at codon 61 of the mouse H-ras gene. The MutEx enrichment is based on MutS binding to a mismatched basepair in heteroduplex DNA. The bound MutS protects the mutant allele from degradation during subsequent exonuclease treatment. ACB-PCR preferentially amplifies a mutant allele in a PCR reaction using a primer that has more mismatches to the wild-type allele than the mutant allele. By combining these two approaches, the codon 61 mutation was detected at mutant fractions as low as 1 in 10(7). This sensitivity was achieved with the thermostable Thermus aquaticus MutS protein but not the Escherichia coli MutS protein. Using the combined approach, the average Pfu DNA polymerase error rate +/- the standard error of the mean for this particular basepair was estimated to be 8 +/- 3 x 10(-7) errors per duplication. The results indicate that MutEx/ACB-PCR is among the most sensitive genotypic selection methods for the detection of mutation.
Parsons
B L
BL
Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, Jefferson, Arkansas 72079, USA. bparsons@nctr.fda.gov
Heflich
R H
RH
D000251:Adenosine Triphosphatases; D000818:Animals; D001426:Bacterial Proteins; D002273:Carcinogens / Q000494:pharmacology; D004252:DNA Mutational Analysis / Q000379:methods*; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D011905:Genes, ras; D005838:Genotype; D051379:Mice; D051719:MutS DNA Mismatch-Binding Protein; D009692:Nucleic Acid Heteroduplexes; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013824:Thermus / Q000378:metabolism
D001426:Bacterial Proteins; D002273:Carcinogens; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009692:Nucleic Acid Heteroduplexes; C072146:Pfu DNA polymerase; D004259:DNA-Directed DNA Polymerase; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli