Stoming T A, Diaz-Chico J C, Yang K G, Efremov D G, Huisman T H
Department of Cell and Molecular Biology, Medical College of Georgia, Augusta 30912-2100.
Hemoglobin. 1988;12(5-6):565-76. doi: 10.3109/03630268808991645.
One of the easiest and most sensitive methods of detecting mutations in the beta-globin gene leading to beta-thalassemia is by the use of oligonucleotide probes. The current method involves digestion of 5-10 micrograms of genomic DNA followed by gel electrophoresis, and blotting onto nitrocellulose. The membrane is then hybridized with a 32P-radiolabeled oligonucleotide probe containing the specific point mutation of interest. Finally, the membrane is subjected to X-ray film for 3-10 days. We wish to report a method for detecting these mutations which involves 1 microgram of genome DNA or less. The method involves the use of a gene amplification technique. A series of primers are synthesized which span the beta-globin gene. In each primer set, one primer is complementary to the beta-gene and the other primer is complementary to the non-coding strand. The suspected mutation point is located between these two primers. With the use of this primer set, the beta-globin gene region is amplified by denaturing, annealing, and DNA synthesis. The amplification cycle is repeated 25 to 30 times. The amplification is conducted using the Klenow fragment of DNA polymerase I or Taq polymerase in the presence of all four deoxynucleotide triphosphates. The resulting amplified DNA is applied to a nylon membrane with the aid of a dot-blot apparatus and directly hybridized with normal and mutant deoxynucleotide probes. The entire process requires one to two days. More than 300 beta-thalassemia homozygotes have been identified in our laboratories; over 20 different mutations have been observed.
检测导致β地中海贫血的β珠蛋白基因突变的最简单且最灵敏的方法之一是使用寡核苷酸探针。当前方法包括将5 - 10微克基因组DNA进行消化，随后进行凝胶电泳，并印迹到硝酸纤维素膜上。然后将该膜与含有感兴趣的特定点突变的32P放射性标记寡核苷酸探针杂交。最后，将膜与X射线胶片接触3 - 10天。我们希望报告一种检测这些突变的方法，该方法仅需1微克或更少的基因组DNA。该方法涉及使用基因扩增技术。合成一系列跨越β珠蛋白基因的引物。在每组引物中，一个引物与β基因互补，另一个引物与非编码链互补。可疑突变点位于这两个引物之间。使用这组引物，通过变性、退火和DNA合成来扩增β珠蛋白基因区域。扩增循环重复25至30次。在所有四种脱氧核苷三磷酸存在的情况下，使用DNA聚合酶I的Klenow片段或Taq聚合酶进行扩增。借助斑点印迹仪将得到的扩增DNA应用于尼龙膜，并直接与正常和突变的脱氧核苷酸探针杂交。整个过程需要一到两天。我们实验室已鉴定出300多名β地中海贫血纯合子；观察到超过20种不同的突变。
