用反向杂交法进行β地中海贫血的产前诊断32例

Direct genotyping and prenatal diagnosis of β-thalassemia mutations by a rapid reverse dot blot assay

本文应用反向点杂交术（ＲＤＢ），进行β地中海贫血的产前诊断。根据中国人地贫的突变基因频率的分布情况，制备两种膜条，一种为“通用”膜条，上面结合了中国人７种常见突变的ＡＳＯ探针；二为“稀少”膜条，结合了检测中国人少见的１１种突变的ＡＳＯ探针。用此方法进行产前诊断３２例，只需一、二次杂交，就能准确区分β地贫不同突变类型的纯合子、杂合子、双重杂合子及正常等位基因。这一技术不依赖同位素、操作简便、检测范围广，可望在我国β地贫的大样本的人群基因调查和临床普及产前诊断起推动作用。

A set of allele specific oligonucleotide(ASO)probes used for detecting all 18 β-thalassemia mutations found in Chinese was immobilized on two strips of Biodyne C membrane;one contains 7 pairs of oligonucleotide probes that are specific for the most commonly found mutant alleles,and the other contains the remaining 11 pairs of ASOs specific for the less commonly found.The membranes were hybridized with β-globin sequences amplified by PCR with biotinylated primers,then treated with Streptavidin-HRP and substrates for color development.The method has been applied successfully to the detection of all 18 Chinese β-thalassemia mutations and the prenatal dingnosis of 32 high risk pregnancies of β-thalassemin.Patients with homozygous, heterozygous and compound heterozygous alleles of these mutations and normal individuals could be easily distinguished by the present method. Using the immobilized- probe format(reverse dot blot),we have been able to screen simultaneously multiple β-thalassemia mutations of a DNA sample by performing hybridization only once.This assay is simple,rapid and independent of radio-isotopes and can be applied for all 18 β-thalassemia mutations so far found in Chinese population.We think that this method may by useful for gene frequency investigation of large nuniber of β-thalassemia DNA samples and used as routine method in the clinic laboratories.