Mutation detection by mismatch binding protein, MutS, in amplified DNA: application to the cystic fibrosis gene.
An experimental strategy for detecting heterozygosity in genomic DNA has been developed based on preferential binding of Escherichia coli MutS protein to DNA molecules containing mismatched bases. The binding was detected by a gel mobility-shift assay. This approach was tested by using as a model the most commonly occurring mutations within the cystic fibrosis (CFTR) gene. Genomic DNA samples were amplified with 5'-end-labeled primers that bracket the site of the delta F508 3-bp deletion in exon 10 of the CFTR gene. The renatured PCR products from homozygotes produced homoduplexes; the PCR products from heterozygotes produced heteroduplexes and homoduplexes (1:1). MutS protein bound more strongly to heteroduplexes that correspond to heterozygous carriers of delta F508 and contain a CTT or a GAA loop in one of the strands than to homoduplexes corresponding to homozygotes. The ability of MutS protein to detect heteroduplexes in PCR-amplified DNA extended to fragments approximately 500 bp long. The method was also able to detect carriers of the point mutations in exon 11 of the CFTR gene by a preferential binding of MutS to single-base mismatches in PCR-amplified DNA.
Lishanski
A
A
Human Genome Center, Lawrence Berkeley Laboratory, University of California, Berkeley 94720.
Ostrander
E A
EA
Rine
J
J
D000251:Adenosine Triphosphatases; D016477:Artifacts; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D003550:Cystic Fibrosis / Q000175:diagnosis / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D016368:Frameshift Mutation / Q000235:genetics; D006579:Heterozygote; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D008967:Molecular Biology / Q000379:methods; D008969:Molecular Sequence Data; D051719:MutS DNA Mismatch-Binding Protein; D009154:Mutation / Q000235:genetics*; D017354:Point Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction; D011485:Protein Binding
D001426:Bacterial Proteins; C505032:CFTR protein, human; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli