用反向点杂交快速检测中国人β—地中海贫血基因突变  

A rapid reverse dot blot assay for all lo β-thalassemia mutations in chinese population

作　　者：张基增 徐湘民[1] 马维芳[1] 单越新[1] 

机构地区：[1]第一军医大学分子生物学实验室

出　　处：《第一军医大学学报》1993年第1期1-5,共5页Journal of First Military Medical University

摘　　要：将一系列针对中国人18种β-地中海贫血(β-地贫)基因突变的ASO探銈固定在尼龙膜条上。用此膜条与用5′端生物素化引物扩增获得的β-珠蛋白基因片段杂交。再用酶标抗生物素蛋白及底物系统检出。用本法检测已知的18种中国人β-地贫突变样品,可准确识别这些突变的杂合子.纯合子和双重杂合子或正常DNA。应用此项技术经一二次杂交即可确定β-地贫基因型。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-地贫的大样本人群基因调查和临床普及产前诊断上起推动作用。A set of allele specific oligonucieotide (ASO) probes used for detecting all 18 β-thalassemia mutations found in Chinese was immobilized on two strips of Biod-yne 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-thalass-emia mutations and the prenatal diagnosis of two high risk pregnancies of β-tha-lassemia.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 be useful for gene frequency investigation of large number of β-thalassemia DNA samples and used as routine method in the clinic laboratories.

关 键 词：地中海贫血 反向点杂交 基因 

分 类 号：R556.610.4[医药卫生—血液循环系统疾病]