White M B, Carvalho M, Derse D, O'Brien S J, Dean M
National Cancer Institute, Frederick Cancer Research and Development Center, Maryland 21702-1201.
Genomics. 1992 Feb;12(2):301-6. doi: 10.1016/0888-7543(92)90377-5.
We have developed a sensitive technique for detecting single base substitutions in polymerase chain reaction (PCR) products from individuals heterozygous for polymorphisms or new mutations. This technique takes advantage of the formation of heteroduplexes in the PCR between different alleles from heterozygous individuals. These heteroduplexes can be detected on polyacrylamide gels because they migrate slower than their corresponding homoduplexes. Using PCR, we have generated a series of point mutations in a defined region of DNA in the equine infectious anemia virus (EIAV). Each mutation is the result of a single base substitution. By mixing the PCR products amplified from these mutations with one another, as well as with wildtype PCR products, we can generate heteroduplexes in which the identity of the mismatched bases is known. We detected eight of nine point mutations using this technique. We have also modified the electrophoretic conditions to optimize the detection of these heteroduplexes. In addition, the usefulness of this technique is demonstrated by its ability to detect a mutation in the cystic fibrosis gene that is the result of a single base substitution. This technique should prove useful for rapidly screening large numbers of individuals for new mutations or polymorphisms.
我们开发了一种灵敏的技术，用于检测来自多态性或新突变杂合个体的聚合酶链反应（PCR）产物中的单碱基替换。该技术利用了杂合个体不同等位基因在PCR中形成异源双链体的特性。这些异源双链体在聚丙烯酰胺凝胶上可以被检测到，因为它们的迁移速度比相应的同源双链体慢。通过PCR，我们在马传染性贫血病毒（EIAV）的特定DNA区域产生了一系列点突变。每个突变都是单碱基替换的结果。通过将从这些突变扩增的PCR产物相互混合，以及与野生型PCR产物混合，我们可以产生已知错配碱基身份的异源双链体。使用该技术，我们检测到了九个点突变中的八个。我们还修改了电泳条件以优化这些异源双链体的检测。此外，该技术能够检测出由单碱基替换导致的囊性纤维化基因突变，证明了其有用性。该技术对于快速筛选大量个体中的新突变或多态性应该是有用的。
