结合多重链置换扩增和等位基因特异性PCR从单细胞中扩增脊髓性肌萎缩症运动神经元生存基因

Combined Multiple Displacement Amplification and Allele-specific PCR to Amplify the Survival Motor Neuron(SMN) Gene from Single Cell

目的:建立结合多重链置换扩增(multiple displacement amplification,MDA)全基因组扩增法和等位基因特异性PCR(allele-specific PCR,AS-PCR)对单细胞进行脊髓性肌萎缩(spinal muscular atrophy,SMA)诊断的方法。方法:对脊髓性肌萎缩症运动神经元生存基因(survival motor neuron gene1,smn1)7号外显子纯合缺失皮肤成纤维细胞及正常的皮肤成纤维细胞使用MDA法进行全基因组扩增,使用AS-PCR检测扩增产物的smn1和smn2基因,同时扩增D5S435、D5S351这2个微卫星位点,评估扩增效率、等位基因脱扣(allele dropout,ADO)率及污染检测。结果:对18个SMA细胞和21个smn1基因正常的细胞进行扩增。smn1和smn2基因扩增成功率分别为90.4%(19/21)和94.8%(37/39)。2个微卫星的扩增成功率为82.1%(32/39)和88.9%(16/18),ADO率分别为16.22%(6/37)和12.5%(2/16)。总扩增成功率为88.89%(104/117),总ADO率为15.09%(8/53)。结论:建立了结合MDA和等位基因特异性PCR对单个细胞进行smn1、smn2基因的扩增方法,为SMA单细胞植入前诊断奠定了基础。

Objective：To develop a single cell diagnosis protocol for spinal muscular atrophy（SMA） based on multiple displacement amplification（MDA） and allele-specific PCR（AS-PCR）.Methods：Genome DNA of single cell of SMA fibroblasts（homozygous deletion of exon7 of survival motor neuron gene 1,smn1） and normal fibroblasts was first amplified by MDA.MDA product was used to amplify the exon 7 of smn1 and smn2 gene using allele-specific PCR,two STR loci D5S435 and D5S351 were amplified simultaneously to evaluate the amplification efficiency and allele dropout（ADO） rate and control the contamination.Results：Eighteen fibroblast cells with homozygous deletion of exon7 of smn1 gene and 20 normal fibroblast cells were amplified.The amplification efficiency of smn1 gene and smn2 gene was 90.48%（19/21） and 94.87%（37/39）,respectively,no smn1 gene was amplified in SMA fibroblast cells.The amplification efficiency for D5S351 and D5S435 was 94.87%（37/39） and 88.89%（16/18）,respectively,the ADO rate for D5S351 and D5S435 was 16.22%（6/37） and 12.5%（2/16）,respectively.The total amplification efficiency was 88.89%（104/117）,total ADO rate was 15.09%（8/53）.Conclusion：A protocol was developed combining with MDA and AS-PCR to analyze smn1 gene and smn2 gene from single cell,which lay the foundation for the PGD of SMA.