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目的:建立多重置换扩增(MDA)联合荧光PCR对植入前胚胎进行性别诊断的方法,为开展性连锁遗传病的性别筛选提供实验依据。方法:在行IVF-ET助孕的患者中,选取废弃的第3日新鲜或冷冻后的2PN受精胚胎。通过显微操作活检得到单个卵裂球细胞,采用MDA方法对单个/2个卵裂球细胞行全基因组扩增,然后采用荧光PCR方法对AMEL、X22、SRY和XHPRT 4个性染色体相关基因或STR位点进行扩增,对其扩增产物行毛细管电泳检测,分析电泳结果确定植入前胚胎的性别。以父母的外周血单个淋巴细胞作为对照进行分析。结果:①采用蛋白酶K法(n=21)和碱法(n=17)对卵裂球细胞进行裂解行MDA,扩增成功率未见统计学差异(85.7%vs 82.4%,P>0.05);单个卵裂球组(n=15)和2个卵裂球组(n=23)MDA扩增成功率亦未见统计学差异(80.0%vs87.0%,P>0.05)。②利用MDA方法对15个胚胎的单个/2个卵裂球进行扩增,扩增成功率为86.7%(n=13)。13个胚胎中7个为男性,6个为女性,性别检测成功率100%;等位基因脱扣(alleledropout,ADO)发生率为7.7%。结论:PGD中MDA是有效且可靠的全基因组扩增方法,MDA结合荧光PCR可以用于性连锁遗传病的性别筛选、致病基因检测和对胚胎进行HLA分型。
Objective: To establish a multi-displacement amplification (MDA) combined with fluorescence PCR for preimplantation embryo sex diagnosis methods to provide experimental evidence for sex-linked genetic disease screening. METHODS: Among the patients who underwent pregnancy with IVF-ET, the rejected 3P fresh or frozen 2PN fertilized embryos were selected. A single blastomere was obtained by microsurgical biopsy. Whole genome amplification was performed on single / 2 blastomere cells by MDA method. Four sex chromosome associated genes (AMEL, X22, SRY and XHPRT) or STR Site amplification, amplification products were detected by capillary electrophoresis, analysis of electrophoresis results to determine the gender of preimplantation embryos. Parents of peripheral blood lymphocytes as a control for analysis. RESULTS: ① The cleavage of MDA by protease K (n = 21) and alkaline (n = 17) did not show any significant difference (85.7% vs 82.4%, P> 0.05) The success rate of MDA amplification in single blastomere group (n = 15) and two blastomere group (n = 23) was also no significant difference (80.0% vs87.0%, P> 0.05). ② The single / double blastomere of 15 embryos was amplified by MDA method. The success rate of amplification was 86.7% (n = 13). Seven out of 13 embryos were males and 6 were females with a 100% success rate for gender detection; the incidence of allele-free alleles (ADO) was 7.7%. CONCLUSION: MDA is an effective and reliable genome-wide amplification method in PGD. MDA-coupled fluorescence PCR can be used to screen sex-linked genetic diseases, detect pathogenic genes and HLA typing of embryos.