简并寡核苷酸引物PCR扩增单细胞基因组的影响因素研究

目的]研究影响简并寡核苷酸引物聚合酶链反应(degenerate oligonucleotide primed PCR,DOP-PCR)扩增单细胞全基因组的因素。方法]以单个人外周血淋巴细胞为材料,进行DOP-PCR,研究新鲜细胞与冷藏细胞及采用新鲜和冻存的细胞裂解物为模板对扩增产物的均匀性及特异性的影响。结果]以新鲜的淋巴细胞为模板与以冷藏的淋巴细胞为模板相比,扩增产物的均匀性相当,特异性上新鲜细胞较好。细胞裂解后立即进行扩增的效果在产物的均匀性及特异性方面均明显优于冻存的细胞裂解物。结论]对单细胞应用DOP-PCR方法进行全基因组扩增时,应尽量选取新鲜细胞进行裂解,裂解后避免冻存以保证扩增的均匀性及特异性。

Study on the Factors Influencing the Whole Genome Amplification from a Single Cell by Degenerate Oligonucleotide Primered PCR

Objective] The purpose was to study the factors influencing the whole genome amplification from a single cell by degenerate oligonucleotide primered PCR(DOP-PCR).Method] With peripheral blood lymphocyte of an individual person as material,the DOP-PCR amplification was conducted to study the influences of using fresh and refrigerated lymphocytes,and fresh and frozen cracking products from lymphocytes as templates on the uniformity and specificity of amplification products.Result] The uniformity of amplification products with fresh lymphocytes as templates was equal to that with refrigerated lymphocytes as templates and their specificity was better.Compared with frozen cracking products from lymphocytes,the effects of immediate amplification after cell cracking were significantly better in the uniformity and specificity of products. Conclusion] When DOP-PCR method was applied in the whole genome amplification from a single cell,it was better to select frech cells for cracking and the frozen storage should be avoided after cracking so as to ensure the uniformity and specificity of amplification.