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


CONCLUSIONS
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.
Reproductive Medicine Center, the First Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang, 325000 People's Republic of China.
Lu-he
LH
Meng
Shi-quan
SQ
Xiao
Xue-feng
XF
Huang
Ying
Y
Zhou
Bing-sen
BS
Xu
D019175:DNA Methylation; D005260:Female; D018392:Genomic Imprinting / Q000235:genetics*; D006801:Humans; D009865:Oocytes / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*