应用巢式PCR对单细胞进行性别诊断的初步研究  

Sex determination of human preimplantation embryo using nested polymerase chain reaction

作　　者：焦泽旭[1] 庄广伦[1] 周灿权[1] 张敏芳[1] 李莉琳[1] 

机构地区：[1]中山大学附属第一医院生殖医学中心,广州510080

出　　处：《中华医学遗传学杂志》2003年第1期64-65,共2页Chinese Journal of Medical Genetics

摘　　要：目的　应用巢式 PCR技术对人类植入前胚胎进行性别诊断。方法　收集单个或两个淋巴细胞和卵裂球 (5 0个 /组 ) ,按不同的方法处理单细胞 (纯水法、冻融法、碱法 ) ,而后行巢式 PCR扩增牙釉质基因。结果　纯水法、冻融法、碱法处理后 ,扩增率分别为 83%、94 %、95 %。后两种处理方法的扩增效率明显高于纯水法 (P<0 .0 1) ,通过检测正常男性单淋巴细胞基因型发现 3种方法等位基因脱失率分别为 2 4 %、12 %、4 % ,差异有显著性 (P<0 .0 5 )。两个淋巴细胞或卵裂球的扩增率及等位基因脱失率与单细胞相比差异无显著性。结论　提高植入前遗传学诊断的准确性主要取决于如何克服单细胞 PCR的缺点 ,采用碱法裂解单细胞及取两个卵裂球进行检测可提高用于性别诊断的单细胞Objective: Using nested polymerase chain reaction (PCR) to perform preimplantation gender diagnosis. Methods: One (or two) lymphocyte and blastomere (n = 50/group) were collected and prepared under the following conditions: (1) water only (H2O); (2) freeze-thaw liquid nitrogen, then boiling; (3) potassium hydroxide/dithiotheriol, heated to 65°C, followed by acid neutralization (KOH). Cells were analyzed by PCR using nested primers amplification with amelogenin gene. Results: The amplification rate and allele dropout (ADO) rate for male lymphocytes by the three methods were 83%, 94%, 95% and 24%, 12%, 4%, respectively. Using two cells per reaction did not increase the amplification rate for the KOH method. Conclusion: The KOH method for DNA preparation is superior to the other methods evaluated. Dual blastomere biopsy and independent blastomere analysis may improve preimplantation diagnostic reliability.

关 键 词：巢式PCR 单细胞 性别诊断 植入前遗传学诊断 胚胎植入 

分 类 号：R446.9[医药卫生—诊断学]