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目的:探讨最适的β地中海贫血疾病HBB基因单细胞全基因组扩增方法。方法:60份β地中海贫血成纤维细胞(HBB基因变异位点CD17和IVSⅡ654)和48份废弃胚胎单个卵裂球进行多次退火环状循环扩增法(MALBAC)和多重置换扩增法(MDA)扩增及高通量测序,比较位点检测率、等位基因脱扣(ADO)率及扩增均一度等。结果:β地中海贫血疾病HBB基因MALBAC技术位点检测率(100%)高于MDA技术(96.3%);CD17和IVSⅡ654的ADO率MALBAC技术为9.09%和0.00%,MDA技术为23.08%和19.23%;对编码人β-珠蛋白的HBB基因附近60个SNP位点检测显示MALBAC技术ADO率为12.04%,MDA技术为21.25%;MALBAC技术拷贝数变异检测变异系数为0.13,MDA技术为0.15。结论:β地中海贫血单细胞诊断MALBAC法优于M D A法。
OBJECTIVE: To explore the single-cell whole genome amplification method of HBB gene which is the most suitable β-thalassemia disease. Methods: Sixty β-thalassemia fibroblasts (HB17, CD17 and IVSⅡ654) and 48 embryos from a single blastomere were used for multiple annealing cycles (MALBAC) and multiple displacement amplification (MDA) ) Amplification and high-throughput sequencing, comparing the detection rate of loci, allele transfer (ADO) rate and amplification uniformity. Results: The detection rate of MALBAC was 100% higher than that of MDA (96.3%) in HBT gene of β-thalassemia disease. The ADO rates of MALBAC and CD17 were 9.09% and 0.009%, 23.08% and 19.23% ; The detection of 60 SNPs in the vicinity of HBB gene encoding human β-globin showed that the ADO rate of MALBAC was 12.04% and that of MDA was 21.25%; the coefficient of variation of MALBAC was 0.13 and that of MDA was 0.15. Conclusion: The single cell diagnosis of β-thalassemia MALBAC is superior to M D A method.