Mutation detection by mismatch binding protein, MutS, in amplified DNA: Application to the cystic fibrosis gene
- Lawrence Berkeley Lab., CA (United States)
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{prime}-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.
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC03-76SF00098
- OSTI ID:
- 86523
- Journal Information:
- Proceedings of the National Academy of Sciences of the United States of America, Vol. 91, Issue 7; Other Information: PBD: 29 Mar 1994
- Country of Publication:
- United States
- Language:
- English
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