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借助PCR(聚合酶链反应)技术可直接测定人基因组DNA中特定基因片段的序列.用双脱氧末端终止法测定单链DNA(ss-DNA)序列比测定双链DNA(ds-DNA)序列效果好,故本实验建立用PCR产生ss-DNA,进而测定其序列以便检测β地中海贫血基因突变的方法.扩增的β珠蛋白基因片段从启动子5’端到第二内含子长615bp.所用引物序列:Ⅰ.5’GCCAAGGACAGGTACGGCT GT CATC 3’;Ⅱ.5’TGC AGC TTG TCA CAG TGC AGC TCA CT 3’.利用PCR产生ss-DNA的策略有两个.一是用引物Ⅰ和Ⅱ扩增出ds-DNA,经1.5％琼脂糖凝胶电泳分离.回收特异的ds-DNA;再以后者
The sequence of a specific gene fragment in human genomic DNA can be directly determined by PCR (Polymerase Chain Reaction) technology, and the effect of double-stranded DNA (ds-DNA) sequence determination by double-deoxy terminator assay Therefore, in this experiment, we established a method to generate ss-DNA by PCR, and then sequenced to detect the mutation of β thalassemia gene.The amplified β-globin gene fragment is 615 bp from the 5 ’end of the promoter to the second intron. Primer sequences used: I.5’GCCAAGGACAGGTACGGCT GT CATC 3 ’; II.5’TGC AGC TTG TCA CAG TGC AGC TCA CT 3’ There are two strategies for generating ss-DNA by PCR: one is to amplify with primers I and II Ds-DNA was added and separated by 1.5% agarose gel electrophoresis, and specific ds-DNA was recovered.