2.3 PCR, cloning and sequencing
Polymerase chain reaction was conducted using motif specific primers
designed for the amplification of MHC class I genes in great reed
warbler. The forward primers HN36 5’-TCCCCACAGGTCTCCACACAGT-3’ and HN46
reverse 5’-ATCCCAAATTCCCACCCACCTT-3’ correspond to exon 3 region in the
flanking introns, the region coding most of the peptide-binding site in
MHC molecules (subunit α2) [22-24]. The primers were used due to
their successful amplification in many passerine species. A list of
primers trialed is given in Table S2. Amplification was performed in the
reaction mixture containing 20ng DNA template,
0.2µM of each
primer, 25µl 2×
EasyTaq® PCR SuperMix (+dye) (Trans, China), and water (deionized) to
reach 50μl as final volume. Thermal cycling for MHC class I
amplification began with one cycle at 94°C for 5 min, followed by 30
cycles of denaturation consisting of sequential steps of 94°C for 30s,
52°C for 30s, and 72°C for 30s, ending with a single extension step at
72°C for 5 min. Purification was carried out with
AxyPrepTMDNA Gel Extraction Kit in accordance with the
manufacturer’s protocol. Purified PCR product was cloned using pEASY
®-T5 Zero Cloning Kit containing Trans1-T1 Phage resistant chemically
competent cells (Transgen Biotech). PCRs were performed for positive
clones using M13 forward and reverse primers. Several colonies (20-25)
per individual were selected and used as a template for sequencing
directionally on an automatic sequencer (ABI PRISM 3730; Invitrogen
Biotechnology Co. Ltd.).