Detection of single-base mutations by a competitive mobility shift assay.
Detection of single-base mutations by a competitive mobility shift assay.
复制标题
通过竞争性迁移位移测定检测单碱基突变。
DOI：
10.1006/abio.1996.0291
复制
发表时间：
1996
影响因子：
2.9
通讯作者：
Hunt,CR
中科院分区：
文献类型：
--
作者：
Chen,M;Laszlo,A;Roti,JL;Hunt,CR
We have developed an assay for the rapid screening of point mutations in specific genes. Our assay is based upon competitive hybridization of differentially labeled wild-type and mutant oligonucleotide probes to a PCR-generated DNA template and a subsequent analysis of the mobility of the probe–template hybrids. The assay is referred to as a competitive mobility shift assay. Generation of a hybridization stringency gradient allows perfect-matched hybrids to be formed to a greater extent at a slightly higher stringency than the corresponding mismatched hybrids. The stringency gradient is achieved by carrying out the hybridizations at a steadily decreasing temperature (from 95 to 20°C) in a thermal cycler. This step allows the assay to be competitive while avoiding the need to establish precise hybridization conditions for each gene-specific probe, a major disadvantage associated with reverse oligonucleotide hybridization. The assay is rapid and sensitive and can selectively detect mutant DNA in the presence of a large (up to one million-fold) excess of wild-type DNA.
