A set of primers was designed for the detection of the FSVs in the
MYH3 gene using available sequence information
(XM_013981330.2). PCR was performed using a Maxime PCR Premix kit, and reactions
included 20 µL of reaction mixture including 100 ng of DNA, 0.5 nmol of
MYH3 promoter-specific primers (MYH3_1_F,
5’-TGGTCTTTCCTAATTGGTGACAT-3’, MYH3_1_R,
5’-AGTTTTGAGCAAGGCTTTTGTT-3’) and distilled water (iNtRON,
Seongnam, Korea). PCR conditions were as follows: initial heating was at
95°C for 5 min, followed by 35 cycles of 30 s for denaturation at
94°C, 30 s for annealing at 65°C, and 30 s for an extension at
72°C, followed by a final extension at 72°C for 10 min in a Nexus
PCR machine (Eppendorf, Hamburg, Germany). And the amplicons were digested with
the restriction enzyme, HpyCH4IV (NEB, Ipswich, MA, USA). The
PCR products were separated on 2.5% agarose gels (Lonza, Basel, Switzerland) and
visualized by UV illumination with a BioFACT 100 bp plus DNA ladder marker
(BioFACT, Daejeon, Korea) [2 (link)].
MYH3 gene using available sequence information
(XM_013981330.2). PCR was performed using a Maxime PCR Premix kit, and reactions
included 20 µL of reaction mixture including 100 ng of DNA, 0.5 nmol of
MYH3 promoter-specific primers (MYH3_1_F,
5’-TGGTCTTTCCTAATTGGTGACAT-3’, MYH3_1_R,
5’-AGTTTTGAGCAAGGCTTTTGTT-3’) and distilled water (iNtRON,
Seongnam, Korea). PCR conditions were as follows: initial heating was at
95°C for 5 min, followed by 35 cycles of 30 s for denaturation at
94°C, 30 s for annealing at 65°C, and 30 s for an extension at
72°C, followed by a final extension at 72°C for 10 min in a Nexus
PCR machine (Eppendorf, Hamburg, Germany). And the amplicons were digested with
the restriction enzyme, HpyCH4IV (NEB, Ipswich, MA, USA). The
PCR products were separated on 2.5% agarose gels (Lonza, Basel, Switzerland) and
visualized by UV illumination with a BioFACT 100 bp plus DNA ladder marker
(BioFACT, Daejeon, Korea) [2 (link)].
