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.).