Pirastu M, Kan Y W, Cao A, Conner B J, Teplitz R L, Wallace R B
N Engl J Med. 1983 Aug 4;309(5):284-7. doi: 10.1056/NEJM198308043090506.
We investigated a method employing synthetic oligonucleotides for the prenatal diagnosis of beta-thalassemia due to a single nucleotide mutation. The beta 0 thalassemia we tested is produced by a nonsense mutation and is commonly found in Sardinia and other parts of the Mediterranean. In this DNA lesion, the glutamine codon CAG at the beta 39 position is mutated to TAG, which results in a stop codon and premature termination of the beta-globin chain. We synthesized two oligonucleotides: one homologous to the normal beta A gene and the other to the beta 0 thalassemia gene at the beta 39 location. The oligonucleotides were labeled with 32P and used as hybridization probes for normal and thalassemic DNA. The beta A probe hybridized only to the normal DNA, and the beta-thalassemia probe only to thalassemic DNA, thus providing a technique for direct demonstration of the mutation. The method is sensitive enough to be applied directly to DNA that is isolated from uncultured cells obtained from only 20 ml of amniotic fluid as early as the 16th gestational week.
我们研究了一种利用合成寡核苷酸对因单核苷酸突变导致的β地中海贫血进行产前诊断的方法。我们检测的β0地中海贫血是由一种无义突变产生的，常见于撒丁岛和地中海其他地区。在这种DNA损伤中，β珠蛋白第39位的谷氨酰胺密码子CAG突变为TAG，导致产生一个终止密码子，使β珠蛋白链提前终止。我们合成了两种寡核苷酸：一种与正常βA基因同源，另一种与β珠蛋白第39位的β0地中海贫血基因同源。这些寡核苷酸用32P标记，并用作正常和地中海贫血DNA的杂交探针。βA探针仅与正常DNA杂交，β地中海贫血探针仅与地中海贫血DNA杂交，从而提供了一种直接证明该突变的技术。该方法灵敏度足够高，早在妊娠第16周时，就可直接应用于从仅20毫升羊水获得的未培养细胞中分离出的DNA。
