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为探索退变寡核甘酸引物多聚酶链反应(DOP-PCR)比较基因组杂交(CGH)单细胞基因组研究的可能性,用DOP-PCR扩增正常XX、XY,异常XO、XXY、13三体和21三体单个细胞基因组DNA,经萤光标记后,分别以正常男性基因组DNA和正常男性单个淋巴细胞DOP—PCR产物为参照,进行CGH分析。结果以基因组DNA为参照的CGH双色荧光强度比均数波动围大,约30％区域的标准差超过正常允许范围,X染色体呈假性过度表达,未能检出13,21三体。以单细胞DOP-PCR产物为参照的CGH强度比均数接近期望值1.0,仅6％的标准差超出正常波动范围,13和21三体的相应染色体大部分常染色质区过度表达。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-CGH及其用于着床前胚胎及母体血胎儿单细胞全基因组研究具有可能性。
In order to explore the possibility of genome-wide hybridization (CGH) single-cell genome research using degenerate oligonucleotide primer-based polymerase chain reaction (DOP-PCR), DOP-PCR was used to amplify normal XX, XY, XO, XXY, 21 trisomy single cell genomic DNA, fluorescently labeled, respectively, the normal male genomic DNA and normal male single lymphocyte DOP-PCR product as a reference for CGH analysis. Results The CGH fluorescence intensity of two CGHs with the reference of genomic DNA fluctuated more than the average. The standard deviation of the 30% region exceeded the normal allowable range. The X chromosome was pseudo-overexpressed and failed to detect the 13,21 trisomy. The CGH intensity averages of single cell DOP-PCR products were close to the expected value of 1.0, with only 6% of the standard deviation outside the normal range of fluctuation, most of the corresponding chromosomes of trisomy 13 and trisomy 21 being overexpressed. CONCLUSIONS: Single-cell DOP-PCR has a nonrandomly inhomogeneous amplification and its effect on CGH can be corrected by using single cell DOP-PCR products as a reference. Single-cell DOP-PCR-CGH and its use in preimplantation embryo and maternal blood fetal single-cell genome-wide research is possible.