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  • Title: [A study on bisulfite sequencing method for methylation status of imprinted genes in single human oocytes].
    Author: Meng LH, Xiao SQ, Huang XF, Zhou Y, Xu BS.
    Journal: Zhonghua Yi Xue Yi Chuan Xue Za Zhi; 2008 Jun; 25(3):289-92. PubMed ID: 18543218.
    Abstract:
    OBJECTIVE: To establish and improve the method of bisulfite 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.
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