引物延伸预扩增结合简并引物PCR在单细胞比较基因组杂交分析染色体异常中的应用

目的 建立一种可信的单细胞全基因组扩增(whole genome amplification.WGA)技术,结合比较基因组杂交(comparative genomic hybridization,CGH)分析单细胞的染色体拷贝数变化,探讨其在胚胎植入前遗传学诊断(preimplantation genetic diagnosis,PGD)中的应用前景.方法 采用引物延伸预扩增结合简并核苷酸引物PCR(primer extension preamplification with degenerate oligonucleotide primed-PCR,PEP-DOP-PCR)的方法,扩增12个已知核型的单细胞标本(包括5个绒毛标本、4个干细胞标本和3个淋巴细胞标本)和4个经PGD检测发现染色体异常的单卵裂球标本,将扩增产物标记红色荧光染料后,与标记绿色荧光染料的正常DNA等量混匀,与正常中期分裂相进行比较基因组杂交分析.同时,应用单纯的简并寡核苷酸引物-PCR(DOP-PCR)扩增10个单细胞DNA,标记后进行CGH分析.比较两种单细胞全基因组扩增方法的扩增效率及随后用于CGH分析染色体拷贝数时的准确性.结果 所有的单细胞采用PEP-DOP-PCR扩增时,均能获得稳定均匀的PCR产物,片段大小范围在100～1000 bp之间,集中分布于400 bp左右的区域,CGH分析结果显示染色体拷贝数变化与其它技术检测的结果一致.而10个单纯的DOP-PCR扩增只有6个标本成功,扩增产物进行CGH分析时,杂交信号不均匀,有2个显示与其它技术分析的结果不一致.结论 PEP-DOP-PCR技术能有效地扩增单细胞的全基因组DNA,其扩增产物可应用CGH技术成功检测单个细胞的染色体拷贝数变化,而单纯的DOP-PCR技术易于出现扩增失败、扩增产物杂交后信号不均一的缺点.PEP-DOP-PCR全基因组扩增结合CGH技术在胚胎植入前遗传学诊断中有良好的应用前景.

Chromosome copy analysis by single-cell comparative genomic hybridization technique based on primer extension preamplification and degenerate oligonucleotide primed-PCR

Objective To establish a single-cell whole genome amplification (WGA) technique, in combination with comparative genomic hybridization (CGH), for analyzing chromosomal copy number changes, and to explore its clinical application in preimplantation genetic diagnosis (PGD). Methods Twelve single cell samples with known karyotypes, including 5 chorionic villus samples, 4 human embryonic stem cell (hESC) samples and 3 peripheral lymphocyte samples, and 4 single blastomere samples carrying chromosomal abnormalities detected by PGD, were collected for whole genome amplification by combining primer extension preamplification (PEP) with degenerate oligonucleotide primed-PCR (DOP-PCR)amplification. The amplified products labeled by red fluorescence were mixed with control DNA labeled by green fluorescence, and then the mixture was analyzed by CGH. As a comparison, 10 single cell samples were amplified by DOP-PCR only and then CGH analysis was performed. Results The amplification using PEP-DOP-PCR was more stable than traditional DOP-PCR. The products of PEP-DOP-PCR range from 100 bp to 1000 bp, with the mean size being about 400 bp. The CGH results were consistent with analyses by other methods. However, only 6 out of 10 single cell samples were successfully amplified by DOP-PCR,and CGH analysis showed a high background and 2 samples showed inconsistent results from other methods. Conclusion PEP-DOP-PCR can effectively amplify the whole genome DNA of single cell.Combined with CGH, this WGA method can successfully detect single-cell chromosomal copy number changes, while DOP-PCR was easy to fail to amplify and amplify inhomogeneousty, and CGH analysis using this PCR product usually showed high background. These results suggest that PEP-DOP-CGH is a promising method for preimplantation genetic diagnosis.