Methylation-sensitive polymerase chain reaction.
Here, we describe a robust and reproducible methylation-sensitive polymerase chain reaction (MS-PCR) method to detect the percentage methylation in repeat sequences of individual pre-implantation ovine embryos produced by different embryo technologies. This method allows the comparison of embryos produced by nuclear transfer with other production and embryo culture methods, accounting for the heterogeneity between embryos within a single treatment. DNA extracted from single embryos is digested with a methylation-sensitive restriction enzyme to determine the percentage methylation after PCR amplification in comparison with an undigested control. The undigested control represents 100% methylation because methylation-sensitive enzymes do not cut methylated DNA, allowing the entire sample to be amplified by PCR. Image analysis quantification of the digested subsample PCR product on an ethidium bromide-stained agarose gel is proportional to the amount of methylated DNA in each embryo. By comparing quadruplicate values obtained for each embryo against a standard curve, we are able to ensure the validity of our results for each individual embryo. Compared with bisulphite sequencing methods, the method described is rapid, inexpensive, and relatively high-throughput.
Moore
Hannah R
HR
Division of Gene Function and Development, Roslin Institute, United Kingdom.
Meehan
Richard R
RR
Young
Lorraine E
LE
D000818:Animals; D001755:Blastocyst / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D019175:DNA Methylation; D004622:Embryo, Mammalian / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D018895:Microsatellite Repeats; D009865:Oocytes / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*; D012756:Sheep
D004247:DNA