The Porcine methylation-specific restriction enzyme PCR (PMP) assay is a
PCR-based methylation method that uses methylation-sensitive restriction
enzymes to determine DNA methylation status. To verify the result of GWBS,
whole blood samples were collected from Berkshire sows to isolate gDNA using
a Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) according
to the manufacturer's instructions and digested with HpaII (NEB,
Hitchin, UK) and MspI (NEB), a pair of methylation-sensitive
isoschizomers that have the same recognition site (CC|GG). An undigested gDNA
(5 g) served as the negative control. Gene-specific primers were
designed to flank the HpaII/MspI sites; these are described
in Table 1. PCR was performed under the following conditions: 94 C
for 5 min, followed by 35 cycles of 94 C for 30 s, 60 C
for 30 s, and 72 C for 30 s. The products were electrophoresed on
a 2 % ( ) agarose gel in 6X loading buffer (Biosesang, Seongnam,
Korea).
PCR-based methylation method that uses methylation-sensitive restriction
enzymes to determine DNA methylation status. To verify the result of GWBS,
whole blood samples were collected from Berkshire sows to isolate gDNA using
a Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) according
to the manufacturer's instructions and digested with HpaII (NEB,
Hitchin, UK) and MspI (NEB), a pair of methylation-sensitive
isoschizomers that have the same recognition site (CC|GG). An undigested gDNA
(5 g) served as the negative control. Gene-specific primers were
designed to flank the HpaII/MspI sites; these are described
in Table 1. PCR was performed under the following conditions: 94 C
for 5 min, followed by 35 cycles of 94 C for 30 s, 60 C
for 30 s, and 72 C for 30 s. The products were electrophoresed on
a 2 % ( ) agarose gel in 6X loading buffer (Biosesang, Seongnam,
Korea).