Diaz-Chico J C, Yang K G, Yang K Y, Efremov D G, Stoming T A, Huisman T H
Department of Cell and Molecular Biology, Medical College of Georgia, Augusta 30912-3331.
Biochim Biophys Acta. 1988 Jan 25;949(1):43-8. doi: 10.1016/0167-4781(88)90052-8.
DNA amplification combined with the use of synthetic oligonucleotide probes has become an important tool in the identification of base substitutions. We report the use of this DNA amplification technique for the detection of mutations in beta-thalassemia. A series of oligonucleotide primers are synthesized which span the beta-globin gene; one primer is complementary to the coding strand and the other to the non-coding strand. The primers are chosen so that there is little homology with other DNA segments, especially the delta gene. Each set of primers spans an area of the gene between 100 and 300 bp, while the suspected mutation point is located between these two primers. With the use of such a primer set, the beta-globin gene region is amplified by denaturation, annealing and DNA synthesis. The amplification cycle is repeated 25-30 times, using the Klenow fragment of DNA polymerase I. The resulting amplified DNA is hybridized with normal and synthetic deoxynucleotide probes using a standard dot-blot method. We have designed a set of primers and experimental conditions which should prove useful to diagnostic centers for detection of numerous beta-thalassemia mutations.
DNA扩增与合成寡核苷酸探针的使用相结合，已成为鉴定碱基置换的重要工具。我们报告了这种DNA扩增技术在检测β地中海贫血突变中的应用。合成了一系列跨越β珠蛋白基因的寡核苷酸引物；一个引物与编码链互补，另一个与非编码链互补。选择引物时，使其与其他DNA片段，尤其是δ基因几乎没有同源性。每组引物跨越基因中100至300 bp的区域，而疑似突变点位于这两个引物之间。使用这样一组引物，通过变性、退火和DNA合成来扩增β珠蛋白基因区域。使用DNA聚合酶I的Klenow片段将扩增循环重复25至30次。使用标准斑点印迹法将得到的扩增DNA与正常和合成脱氧核苷酸探针杂交。我们设计了一组引物和实验条件，这对诊断中心检测多种β地中海贫血突变应是有用的。
