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目的:应用并评价多次退火环状循环扩增(multiple annealing and looping-based amplification cycles,MALBAC)技术在单细胞水平诊断脊髓性肌萎缩症(spinal muscular atrophy,SMA)基因变异的效率。方法:收集SMN1基因7号外显子纯合缺失、正常皮肤成纤维细胞以及废弃胚胎单个卵裂球细胞,分别使用MALBAC和多重链置换扩增(multiple displacement amplification,MDA)方法进行全基因组扩增(WGA)。Sanger测序检测SMN1及SMN2序列,并对3个微卫星位点进行连锁分析。结果:2种扩增技术的总扩增成功率、等位基因脱扣(ADO)率无统计学差异(P>0.05);MALBAC组诊断准确率为91.7%(67/73),低于MDA组的96.1%(73/76)(P<0.05)。结论:针对SMA疾病开展单细胞水平遗传学诊断,传统MDA方法略优于MALBAC全基因组扩增技术。
OBJECTIVE: To evaluate and evaluate the efficiency of multiple annealing and looping-based amplification cycles (MALBAC) in the diagnosis of spinal muscular atrophy (SMA) gene mutation at single cell level. Methods: The homozygous deletion of SMN1 gene exon 7, normal skin fibroblasts and single embryonic blastomere were collected. The whole genome amplification was performed using MALBAC and multiple displacement amplification (MDA) WGA). Sanger sequencing detected SMN1 and SMN2 sequences, and analyzed the linkage of three microsatellite loci. Results: The success rates of total amplification and allele release (ADO) of the two kinds of amplification did not reach statistical significance (P> 0.05). The diagnostic accuracy of MALBAC was 91.7% (67/73) 96.1% (73/76) of the group (P <0.05). CONCLUSION: Single cell-level genetic diagnosis of SMA disease is slightly superior to the traditional MDA method over MALBAC genome-wide amplification.