PCR方法对单细胞SRY基因扩增效率的对比研究

目的探讨单细胞基因扩增时,巢式PCR和引物预扩增(PEP)-巢式PCR两种扩增方法对SRY基因脱扣的影响程度;并应用PEP-巢式PCR方法对单个卵裂球细胞进行SRY基因诊断。方法获取单个正常男女淋巴细胞,随机分为巢式PCR组和PEP-巢式PCR组。同时扩增SRY基因和ZP3基因位点。选用IVF-ET后冻存的4个胚胎,处理后获取单个卵裂球11个,用PEP-巢式PCR扩增,鉴定其性别。结果单个淋巴细胞经巢式PCR和PEP-巢式PCR方法扩增后,其基因扩增成功率分别为92.39%,98.91%;性别诊断正确率分别为86.00%,98.00%;SRY基因的脱扣率分别为16.67%,2.38%。两者有统计学检验均有显著性差异(P<0.05。对单个卵裂球应用PEP-巢式PCR方法进行SRY基因和ZP3基因扩增后,有3个胚胎的8个卵裂球被诊断为男性,而另外1个胚胎的3个卵裂球被诊断为女性。结论1.行单细胞基因扩增时,PCR扩增方法会影响等位基因脱扣的发生率。2.对性连锁遗传病进行植入前遗传学诊断时,采用PEP-巢式PCR方法扩增SRY基因和ZP3基因对单个细胞对进行性别鉴定时,可以有效地降低SRY基因脱扣的发生率,提高性别诊断的特异性和敏感性,能够用于单细胞的性别诊断,可用于性连锁遗传病的植入前遗传学诊断。

Comparsion of nested polymerase chain reaction and primer extension preamplification for single-cell sexing

Objectives: To evaluate nested PCR and PEP-nested PCR in a single cell to determine the ability to accurately amplify and correctly diagnose a targeted gene, and to apply PEP-nested PCR to sexing in a single pre-embryo blastomere. Methods: 84 male and 16 female single lymphocytes were collected and divided randomly into two groups: a: amplified SRY and ZP3 gene by nested PCR . b: amplified whole genome by 15-base random primers(PEP), then a small aliquots of PEP product were re-amplified SRY and ZP3 gene by nested PCR. The ADO rate of SRY, amplification rate and correct diagnosis rate of two amplifications were analyzed with exect method. 11 single blastomeres from 4 embryos were collected and PEP-PCR in order to diagnose theirs sex. Results: After biopsied single lymphocytes amplificed with nested PCR and PEP-nested PCR, the allele dropout (ADO) rate of SRY gene of theirs were16.67%, 2.38%, while amplification rate were 92.39%, 98.91%; correct diagnosis rate were 86.00%, 98.00%, respectively. After blastomeres amplificed by PEP-nested PCR, 8 blastomeres from 3 pre-embryo were diagnosed male, whereas 3 from the other were female. Conclusion: On condition that a single cell was amplificed, amplification methods can influence the allele dropout rate. When the preimplantation genetic diagnosis of X-linked disease was conducted, PEP-nested PCR can reduce the allele dropout rate of SRY gene, and raise the diagnostic sensitivity and specifity . It is suitable for the preimplantation genetic diagnosis of X-linked disease,and other single gene hereditary diseases.