MUTATION DETECTION BY MISMATCH BINDING-PROTEIN, MUTS, IN AMPLIFIED DNA - APPLICATION TO THE CYSTIC-FIBROSIS GENE

被引:54
作者
LISHANSKI, A [1 ]
OSTRANDER, EA [1 ]
RINE, J [1 ]
机构
[1] UNIV CALIF BERKELEY,DEPT MOLEC & CELL BIOL,BERKELEY,CA 94720
关键词
PCR AMPLIFICATION; DNA HETERODUPLEXES; DNA-BINDING PROTEIN; MOBILITY-SHIFT ASSAY;
D O I
10.1073/pnas.91.7.2674
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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 DELTAF508 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 DELTAF508 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 almost-equal-to 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.
引用
收藏
页码:2674 / 2678
页数:5
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