Lukacs et al. BMC Genomics 2010, 11:154 http://www.biomedcentral.com/1471-2164/11/154 Page 13 of 17 Methods BAC library screening An Atlantic salmon CHORI-214 bacterial artificial chro- mosome (BAC) library was obtained from BACPAC Resources, Children’s Hospital Oakland Research Insti- tute [36]. The library consisted of approximately 299,000 recombinant clones, representing 18-fold gen- ome coverage and an average insert size of 188 kb [37]. Putative full length cDNA sequence information for the S. salar SAA, UHA and ZE genes were obtained from GRASP EST clustering database [26,27] and used for primer design. Probes specific for the SAA, UHA and ZE genes were PCR amplified (primers listed in Table 3) from a Head Kidney cDNA pool, purified from agarose gel slices with the GeneClean III Kit (Qbiogene) and verified by sequencing. Probes were radioactive labelled with a32P-CTP (Amersham) using Rediprime Random Labelling Kit (Amersham), including spermine precipita- tion of labelled DNA. Filter hybridizations were con- ducted as described by CHORI. Probed BAC library filters were stored in Phospho-image cassettes for 2-24 hours and hybridizations visualized by a Typhoon Phos- pho Image Scanner (Amersham). Characterization of BACs SAA, UHA and ZE positive BAC clones were ordered into contigs by GRASP HindIII fingerprint information [38]. Selected BAC clones were picked from each Table 3 Primers used for probes, screening and real time PCR Sequence (5’-3’) SAA_387F1 TATGAGCCATGCATATGAC SAA_815R1 CTGTAGCTCTGGGTGTCCT UHA_329F1 TGCAGAAAATGTACAGCTG UHA_798R1 GTAGTTGTGCTGCTGTAGG ZE_481F1 TTGAAATTCATAAAGGGCAC ZE_909R1 GGGATCTGCACACTCATC 114I13-F2 TGATTCCCATCTCAGTATCC 114I13-R TAGCTACCTTTCTGAGCCTG 184H23-F2 CAGGCCTTTACTCTCTGCTA 184H23-R GGGAGTGTTGTCTAAACCTG 21M13 TGTAAAACGACGGCCAGT UBA-F CTGACAACTCTGGGAGAGCT UBA-R GTGTGTTATGTTCTTGAGACGT 439_UDA_967F CCATCATTGTCCCCATCATTG 439_UDA 1094R AGATCTTTCCCAGAGTACACA UGA-F GTGAATGATCAGGTCATTAGCC UGA-R CTCTGCGCCTCTCAGGTCC UHA1_800F TACAGCAGCACAACTACACAT UHA1_920R TGACCAATACCAAGACCATGTAGA UHA2end-F TACACATGCACTGTCCAACAC UHA2end-R GCCAATGTGACCAATACAATGG ULA_961F TCAAGACCAACTGGGGAAACA ULA_1094R CCGTCTTTCTTCTCTTGTCTG ZAA_847F CCAGAGGCAGACAAGGAAAC ZAA 954R CAGCCCCAATGACTACAGCA ZBA_840F CCCAGAGGCAGACAAGGACA ZBA_949R AATTAGAGAGGCCTGGATCCC ZCA_842F CAGAGGCAGACAAGCAAACC ZCA_963Rseq ATTGATAACCATGCAAATAACTGG SAA_379F CCTTTATGAGCCATGCATATGA SAA_504R AATGAATGACCAGCCTAACAAG Usage cDNA amplification of SAA cDNA amplification of SAA cDNA amplification of UHA cDNA amplification of UHA cDNA amplification of ZE cDNA amplification of ZE Marker Ssa10083BSFU in BAC 114L13 Marker Ssa10083BSFU in BAC 114L13 Marker Ssa10084BSFU in BAC 184H23 Marker Ssa10084BSFU in BAC 184H23 Realtime PCR Sasa-UBA Realtime PCR Sasa-UBA Realtime PCR Sasa-UDA Realtime PCR Sasa-UDA Realtime PCR Sasa-UGA Realtime PCR Sasa-UGA Realtime PCR Sasa-UHA1 Realtime PCR Sasa-UHA1 Realtime PCR Sasa-UHA2 Realtime PCR Sasa-UHA2 Realtime PCR Sasa-ULA Realtime PCR Sasa-ULA Realtime PCR Sasa-ZAA Realtime PCR Sasa-ZAA Realtime PCR Sasa-ZBA Realtime PCR Sasa-ZBA Realtime PCR Sasa-ZCA Realtime PCR Sasa-ZCA Realtime PCR Sasa-SAA Realtime PCR Sasa-SAA 1 Primer design for probes for SAA, UHA and ZE is based on DW536070, DW548896 and DQ099914 respectively. 2 The forward primer had the 21M13 primer added to its 5’-end.