CAJ | 학술논문

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H19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.
목적 건립화개진채용아류산경납측서검측단개인란자인기기인갑기화상태적방법.방법 대초촉배란래원적인류단개란자용저용점경지당포매,아류산경납처리후행PCR확증,PCR산물극륭측서학정인기기H19화MEST적CpG위점적갑기화상태.결과 재개진료저용점경지당포매화아류산경납처리방법후,단개란자적PCR성공솔체도82.46%,체세포오염솔위7.14%.극륭측서결과현시,기서렬부합이론서렬,서렬중미견비CpG적포밀정.결론 단개란자아류산경납측서검측인기기인갑기화상태적방법구유고효、은정적특점,가이위진일보연구인류보조생식기술대기인인기적영향제공기출.
Objective To establish and improve the method of bisulfite sequencing for methylation status of imprinted genes in single human oocytes.Methods Single supemvulated immature human oocyte was embedded into lowmelting point agarose.followed by bisulfite treatment and Oolymerase 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 modifled methods of emhedding 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.Condusion The bisulfite sequencing method we used to deter-mine the methylation slatus of imorinted 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.