亚硫酸氢钠测序检测单个人卵子印记基因甲基化状态方法的改进

A study on bisulfite sequencing method for methylation status of imprinted genes in single human oocytes

目的建立和改进采用亚硫酸氢钠测序检测单个人卵子印记基因甲基化状态的方法。方法对超促排卵来源的人类单个卵子用低熔点琼脂糖包埋，亚硫酸氢钠处理后行PCR扩增，PCR产物克隆测序确定印记基因1-119和MEST的CpG位点的甲基化状态。结果在改进了低熔点琼脂糖包埋和亚硫酸氢钠处理方法后，单个卵子的PCR成功率达到82．46％，体细胞污染率为7．14％。克隆测序结果显示，其序列符合理论序列，序列中未见非CpG的胞嘧啶。结论单个卵子亚硫酸氢钠测序检测印记基因甲基化状态的方法具有高效、稳定的特点，可以为进一步研究人类辅助生殖技术对基因印记的影响提供基础。

Objective To establish and improve the method of bisalfite sequencing for methylation status of imprinted genes in single human oocytes. Methods Single superovulated immature human oocyte was embedded into low melting point agarose, followed by bisulfite treatment and polymerase chain reaction （PCR） amplification of the H19 and MEST genes. The PCR products were then subjected to TA cloning and sequencing to determine the methylation status. Results With the modified methods of embedding and bisulfite treatment, we achieved a high PCR success rate of 82.46%, with the somatic cell contamination rate as low as 7.14%. The sequencing results showed no non-CpG cytosine and exact conformity to the theoretical sequences. Conclusion The bisulfite sequencing method we used to determine the methylation status of imprinted genes at the single-cell level was highly efficient and reliable, which can serve as a foundation for the further study of the influences of human assisted reproductive technology on genomic imprinting.