单细胞水平β地中海贫血基因突变检测技术的实验研究  

The study of detecting β-thalassaemia mutations from a single cell.

作　　者：易萍[1] 李力[1] 邓兵[2] 姚宏[1] 陈竹钦[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所妇产科,400042 [2]重庆医科大学附属儿童医院,400014

出　　处：《中国优生与遗传杂志》2005年第4期19-21,共3页Chinese Journal of Birth Health & Heredity

基　　金：国家科技部 973项目 (2 0 0 1CB5 10 3 0 5 )

摘　　要：目的　探讨在单个淋巴细胞水平能否同时检测β地中海贫血多个突变。方法　将我国β地中海贫血常见8种突变点(CD4 1- 4 2、IVS -Ⅱ- 6 5 4、CD17、TATAboxnt - 2 8、CD71- 72、TATAboxnt - 2 9、CD2 6、IVS -Ⅰ- 5 )的等位特异性寡核苷酸(allelespecificoligonucleiotide ,ASO)探针点样于尼龙膜上,制备β地中海贫血常见突变诊断膜条。应用蛋白酶裂解法处理单个细胞,采用15个碱基的随机引物对单个细胞的基因组进行引物延伸预扩增(primerextensionpreamplification ,PEP) ,取其产物5 μl分别对β地中海贫血的目的基因片段进行巢式或半巢式PCR扩增与生物素标记,然后采用β地中海贫血常见突变诊断膜条与标记产物进行反向斑点杂交(reversedotblot,RDB)检测β地中海贫血突变型。结果　使用显微操作法分别从1例正常女性及4例β地中海贫血血标本中获得3个淋巴细胞。采用Rechitsky等的蛋白酶K裂解法处理单个淋巴细胞,扩增效率为93.3% ,等位基因脱扣率为6 .7%。RDB杂交后结果显示与预料的相符,其基因型依次为N/N、CD17(A→T) /N、IVS-Ⅱ- 6 5 4 (C→T) /CD17(A→T)、CD4 1- 4 2 (-CTTT) /N、TATAboxnt - 2 8(A→G) /N。结论　在单细胞水平应用PEP及RDB技术可以同时检测β地中海贫血多个突变,操作相对简便,为胚胎植?Objetive: To explore a technology for detecting β-thalassaemia multipoint mutations from a single cell simultaneously. Methods: A set of allele specific oligonucleotide (ASO) probes used for detecting the eight familiar β-thalassaemia mutations (CD41-42, IVS-Ⅱ-654, CD17, TATA box nt -28, CD71-72, TATA box nt -29, CD26, IVS-Ⅰ-5) were immobilized on a strip of nylon membrane. The genome of a individual cell was amplified by primer extension preamplification (PEP) with the mixture of 15-base random oligonucleotides. The aliquots (5μl) from PEP were used to amplify the objective gene fractions of β-thalassaemia gene by nested or semi-nested PCR. The membrane was hybridized with the final amplified products and then treated with Streptavidin - HRP and color development. Results: 3 lymphocytes were picked up from blood samples of 1 healthy female and 4 patients with known β-thalassaemia mutations respectively. Each single lymphocyte was lysed in the proteinase K buffer. The amplification efficacy was 93.3% and alle drop-out rate was 6.7%. RDB was applied to the final amplified products from the five participants. The results of diagnosis were same to the expected, and their genotypes were N/N, CD17(A→T)/N, IVS-Ⅱ-654(C→T)/CD17(A→T), CD41-42(-CTTT)/N and TATA box nt -28(A→G)/N, respectively. Conclusion: PEP and RDB could detect multiple β-thalassaemia mutations from a single cell simultaneously and be applied to preimplantation genetic diagnosis and non-invasive prenatal diagnosis for β- thalassaemia.

关 键 词：单细胞 Β地中海贫血 基因突变 检测技术 实验研究 

分 类 号：R446.9[医药卫生—诊断学]