单细胞退变寡核苷酸引物PCR-比较基因组杂交研究

Single cell degenerate oligonucleotide primer PCR and comparative genomic hybridization

目的 探索单细胞退变寡核甘酸引物PCR(DOP-PCR) -比较基因组杂交 (CGH)技术应用于单细胞全基因组分析及着床前胚胎遗传学研究的可能性。方法 建立单个淋巴细胞的DOP -PCR技术 ,分别以正常男性的组织DNA和单细胞DOP -PCR产物为参照 ,进行正常男、女性和X单体、X、13和21三体单细胞DOP-PCR产物的CGH。结果 X染色体在以组织DNA为参照的CGH中呈假性过度表达,13、21三体未能显示阳性结果。以单细胞DOP -PCR产物为参照的CGH不仅能正确反映X和Y拷贝数 ,而且能显示13和21三体相应染色体的过度表达。结论 单细胞DOP-PCR存在非随机的不均匀扩增 ,其对CGH的影响 ,能通过应用单细胞DOP -PCR产物为参照而得以纠正。单细胞DOP-PCR

Objective To investigate the possibility of applying degenerate oligonucleotide primer PCR(DOP-PCR) and comparative genomic hybridization(CGH) in analysing genomic genetics of a single cell.Me-thods Amplification of the whole genomic DNA of a single cell with XX,XY,XO,XXY,+13 or +21 by DOP-PCR.CGHs of a single cell DOP-PCR product against the genomic DNA and a single cell DOP-PCR product from normal male were carried out respectively.Results The mean fluorescence intensive ratio in CGH with the genomic DNA as reference was greatly fluctuated and the standard deviation in about 30% haploid was beyond the normal limits.False positive hyperepresentation was existed in X chromosome while trisome 13 and 21 failed to be detected.However,the distributions of the mean and the standard deviation of the ratio in the CGH with DOP-PCR product as reference were quite acceptable.The copy number changes of chromosome X?Y?13 and 21 were demonstrated.Conclusions There is unrandom unequal amplification in a single cell DOP-PCR.Using a single cell DOP-PCR product as reference can decrease its influence on CGH.Single cell DOP-PCR-CGH and its application in preimplantation embryo or fetal cell in maternal blood may be possible.