论文部分内容阅读
从1例广东籍重型β地贫患儿的外周血白细胞中分离基因组DNA。回收用HindⅢ完全酶解产生的6～9kb长度的DNA片段,与λCharon 28噬菌体DNA插入重组,构建基因文库。用~(32)P标记的βIVSⅢ(0.9kb)DNA片段作为探针,从1.4×10~5噬菌斑中筛出3个带有β地贫基因的阳性克隆,对其中一个阳性重组体DNA进行限制酶谱分析,进一步分离得到带有β地贫基因的3.7kb DNA片段,与噬菌体M13mp19重组,采用斑点杂交法筛选出阳性次级克隆。制备阳性M13mp19单链重组体DNA,用作DNA序列分析。在国内完成了首例β地中海贫血基因的克隆化分离。
Genomic DNA was isolated from peripheral white blood cells in 1 case of severe β-thalassemia from Guangdong. A DNA fragment of 6-9 kb in length, completely digested with HindIII, was recovered and inserted into λCharon 28 phage DNA to construct a gene library. Three ~ (32) P-labeled βIVSⅢ (0.9kb) DNA fragments were used as probes to screen out 3 positive clones with β thalassemia gene from 1.4 × 10 ~ 5 plaques. One positive recombinant DNA Restriction enzyme digestion analysis was performed to further isolate the 3.7 kb DNA fragment with β-thalassemia gene and recombine with phage M13mp19. The positive clones were screened by dot blot hybridization. Positive M13mp19 single-stranded recombinant DNA was prepared for DNA sequence analysis. In China, the first β-thalassemia gene was cloned and isolated.