Detection of single DNA base mutations with mismatch repair enzymes.
A novel method for identifying DNA point mutations has been developed by using mismatch repair enzymes. The high specificity of the Escherichia coli MutY protein has permitted the development of a reliable and sensitive method for the detection and characterization of point mutations in the human genome. The MutY protein is involved in a repair pathway that can convert A/G or A/C mismatches to C/G or G/C basepairs, respectively. A/G or A/C mismatches formed by hybridization between two amplified genomic DNA samples or between specific DNA probes and target DNA are nicked at the mispaired adenine strand by MutY protein. As little as 1% of the mutant sequence can be detected by the mismatch repair enzyme cleavage (MREC) method in a mixture of normal and mutated DNAs (e.g., mutant cells are only present in 1% of the normal cell background). By using different probes, the assay also can determine the nucleotide sequence of the mutation. We have applied this method to detect single-base substitutions in human oncogenes.
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Department of Biological Chemistry, School of Medicine, University of Maryland, Baltimore 21201.
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D001483:Base Sequence; D045647:DNA Glycosylases; D004252:DNA Mutational Analysis / Q000379:methods*; D015342:DNA Probes; D006801:Humans; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D009447:Neuroblastoma; D009841:Oligonucleotides / Q000138:chemical synthesis; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D015342:DNA Probes; D009841:Oligonucleotides; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C061738:mutY adenine glycosylase