全基因组扩增在DMD植入前遗传学诊断中的可行性研究

目的:建立单细胞扩增前引物延伸法及巢式PCR技术,研究该技术在杜氏肌营养不良症植入前遗传学诊断中的可行性。方法:获取单个正常男女淋巴细胞和无杜氏肌营养不良(DMD)家族史的单个卵裂球,15碱基随机引物PEP扩增单细胞全基因组,再以巢式PCR技术检测PEP反应产物中DMD基因外显子8,17,19,44,45,48及Y染色体上的睾丸决定基因SRY。结果:单个淋巴细胞和单个卵裂球的6个DMD外显子扩增成功率分别为97.2％(175/180)和100％(60/60),单个男性淋巴细胞SRY的扩增成功率为100％(15/15),而单个女性淋巴细胞无SRY的扩增(0/15)。结论:检测单细胞DMD基因外显子8,17,19,44,45,48和SRY的PEP-巢式PCR技术具有较高扩增成功率和特异性,可望应用于 DMD的植入前遗传学诊断的单细胞基因诊断中。

Single Cell Analysis of DMD Gene and SRY Gene Using Whole Genome Amplification .

Objective:To investigate a reliable and sensitive method for the detection of sex and multi-loci of duchenne muscular dystrophy(DMD)gene in single cell. Methods:Whole genome of single cell were amplified using 15-base random primers (Primer extension preamplification,PEP),then a small aliquot of PEP product were analyzed by locus-specific nest PCR amplification. The procedure was e-valuated by the detection of dystrophin exons 8,17,19,44,45,48 and human testis-determining gene or detecting (SRY)in single lymphocyte from known sources and single blastomer from the couples with no family history of DMD. Result : The amplification efficiency of six dystrophin exons from sin-gle lymphocyt and single blastomere are 97.2%(175/180)and 100%(60/60) respectively. SRY showed 100% (15/15) of amplification in single male-derived lymphocyt and 0%(0/l5) of amplifica-tion in single female-derived lymphocyt. Conclution: The technique of single cell PEP-nest PCR for the determinatin of dystrophin exons 8,17,19,44,45,48 and SRY is highly successful and specific. PEP-nest PCR is suitable for the preimphantation genetic diagnosis (PGD) of DMD at single cell lev-el.