Evaluation of MutS as a tool for direct measurement of point mutations in genomic DNA.
The MutEx assay is a technique that was developed to detect and map mutations. This assay takes advantage of the Escherichia coli mismatch binding protein MutS, which binds and protects mismatched, heteroduplex DNA from subsequent exonuclease digestion. The plausibility of using the MutEx assay as part of a genotypic selection scheme was investigated. Heteroduplexes were formed between mouse H-ras gene PCR products or restriction fragments that contained wild-type sequence and sequence with a single base change at codon 61 (wild-type, CAA and mutant, AAA). The heteroduplexes were incubated with MutS and then treated with the exonuclease activity of T7 DNA polymerase. MutS-protected DNA sequences were amplified by PCR. When this method was linked to single nucleotide primer extension (SNuPE) for mutant base identification, original mutant fractions of 1 in 50000 and above were detected. Using comparable DNA template mixtures, the sensitivity of SNuPE alone was 1 in 5 or 1 in 50, depending on the direction of SNuPE priming and the particular base being incorporated. We conclude that the MutEx assay was able to enrich the mutant sequence approximately 1000-fold and, therefore, has considerable potential as a tool for mutation detection.
Parsons
B L
BL
Division of Genetic Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, USA. bparsons@nctr.fda.gov
Heflich
R H
RH
D000251:Adenosine Triphosphatases; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D017931:DNA Primers; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D011905:Genes, ras / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D051719:MutS DNA Mismatch-Binding Protein; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism*; D009693:Nucleic Acid Hybridization; D017354:Point Mutation; D016133:Polymerase Chain Reaction
D001426:Bacterial Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009692:Nucleic Acid Heteroduplexes; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli