H19差异甲基化位点MSRE-qPCR分析识别精子DNA新方法的可行性探究

Application of H19 ICR MSRE-qPCR for sperm DNA identification

目的:研究位于H19转录起始点-3390 bp的HhaI位点是否为差异甲基化位点,为精子DNA识别提供新依据。方法:从51份血样和20份精子样本提取基因组DNA,每个样本分别在同一反应体系中进行甲基化敏感酶HhaI消化和荧光定量PCR反应(MSRE-qPCR),扩增包含上述HhaI位点的114 bp片段。得到每个样本酶切前后的Ct值,用2^(-△△Ct)计算各样本酶切前后模板DNA相对表达量,将其作为DNA甲基化水平。结果:51份血样中该HhaI位点甲基化水平为(44.4±23.8)%,20份精子DNA甲基化水平为(120.5±52.5)%,差异有统计学意义(P<0.001)。结论:甲基化水平高于94%可识别精子DNA,H19差异甲基化区114 bp序列的MSRE-qPCR分析为精子DNA的识别提供了一种新方法。

Objectives： To study whether HhaI site,located at the transcription start point- 3390 bp of H19 gene,was differentially methylated site,to provide basis for sperm DNA identification. Methods： These genomic DNA were extracted from 51 blood samples and 20 semen samples,and then every DNA sample was subjected to digestion by a methylationsensitive restriction endonuclease HhaI and qPCR in the same reaction system to amplify the 114 bp target fragment which contained the Hha I site. The Ct of digested group and undigested group were determined from each sample. The DNA methylation level was described by 2^（-△△Ct）. Results： DNA methylation level in this HhaI site was 44. 4 % ± 23. 8% in 51 blood samples and 120. 5% ± 52. 5% in 20 semen samples. The difference was of statistical significance（ P ﹤ 0. 001）. Conclusion： Sperm DNA could be identified if H19 HhaI site methylation level is over 94%. MSRE- qPCR analysis on 114 bp H19 differentially methylated regions is a newmethod to identify sperm DNA.