Long-range PCR: primer design and reaction conditions

With the aim of capturing DRB copies that may have passed undetected in our previous study (Mona et al. 2008), we designed primers on DRB conserved regions, putatively shared by multiple copies of the locus (Fig. ​(Fig.1).1). Exons 1, 2, and 3 nucleotide sequences were compared within and between species to estimate their level of conservation. The proportion of variable nucleotide positions per exon within B. taurus DRB calculated from the Ensembl database (www.ensembl.org) is 0.06 for exon 1 and 0.11 for exon 3, while it increases to 0.21 for exon 2. Between Bovidae species (Table S1a, median time of divergence between chamois and cattle: 24.60 MYA, interval 21.41–29.46 MYA, www.timetree.org) the proportion of variable sites becomes 0.19, 0.26, and 0.09 for exons 1, 2, and 3, respectively. Given the higher proportion of conserved sites, primers were designed on exons 1 and 3. This approach required to set-up a long-range PCR reaction for a range of length between 9 Kb (based on O. aries GenBank {"type":"entrez-nucleotide","attrs":{"text":"AM884914","term_id":"165931963","term_text":"AM884914"}}AM884914) and 16 Kb (based on B. taurus Ensembl: ENSBTAG00000013919). The intron length of chamois’ DRB was unknown, since only the cDNA had been deposited in GenBank ({"type":"entrez-nucleotide","attrs":{"text":"AF336340.1","term_id":"18033488","term_text":"AF336340.1"}}AF336340.1). The inclusion of intron 1, exon 2, and intron 2 was expected to capture the most variable part of the locus.

Long-PCR primers located in exons 1 and 3 were designed using Primer3 (biotools.umassmed.edu/bioapps/primer3_www.cgi) and considering (1) B. taurus DRB polymorphic sites available in Ensembl database, (2) conserved regions in the alignment between orthologous DRB loci from 15 different Bovidae species (Table S1a), and (3) DRB-specific regions resulting from the alignment with the paralogue DQB from B. taurus and O. aries, showing a high degree of sequence similarity (Table S1b, Supporting information for accession numbers). The primer pair DRB_Ex1_Fwd1 5′-GATGCTGAGCCCTCCCCTGG-3′ and DRB_Ex3_Rev2 5′-CCGGAACCACCTGACTTCAA-3′ was used for the DRB long-range PCR (Fig. ​(Fig.11).

Long-range PCR conditions were as follows: the 50-µl reaction mixture contained 1 U of Platinum Taq DNA Polymerase High Fidelity (Life Technologies), 1X buffer HiFi, 2 mM Mg(SO)₄, 0.2 mM each dNTP, 0.2 µM each primer, and 50–100 ng of genomic DNA. The thermal cycling profile included an initial denaturation at 94 °C for 1 min followed by 35 cycles of 94 °C for 20 s, 62 °C for 30 s, 68 °C for 15 min. The PCR product was run on 1% low melting SeaPlaque™ GTG™ Agarose gel; the expected ~9 Kb band was cut and extracted using the MinElute Gel Extraction Kit (QIAGEN). Both long-PCR products and fragments extracted from the gel were run on a Bioanalyzer 2100 (Agilent) using the DNA 12000 chip assay.