Universal PCR primers for ribosomal protein gene introns of fish

Abstract

Human ribosomal protein (RP) gene sequences with respect to intron/exon structures and corresponding cDNA or genomic data of fish species were obtained from the GenBank database. Based on conserved exon sequences, 128 primer pairs for 41 genes were designed for exon-primed intron-crossing (EPIC) polymerase chain reaction (PCR). In reference to the draft genome sequences of the Pacific bluefin tuna (Thunnus orientalis), 12 primer pairs expected to amplify introns of the bluefin tuna with lengths of 500-1000 bp were selected and applied to six distantly related fish species belonging to the Orders Clupeiformes, Tetraodontiformes, Pleuronectiformes, Perciformes, Scorpaeniformes, and Anguilliformes. PCR amplification was observed for at least four species in each primer pair, and all fragments were larger than those expected for intronless amplification. Single fragment amplification was observed for at least seven primer pairs per species. Fragment sizes of the bluefin tuna for nine primer pairs corresponded to those expected from the genomic data. Thus, our primer pairs are potentially applicable to a wide variety of fish species and serve as an initial step for isolating single-copy nuclear DNA sequences.

Universal primers for exon-priming intron-crossing (EPIC) PCR on ribosomal protein genes in marine animals

“…Exon-primed intron-crossing (EPIC) polymerase chain reaction (PCR) has been developed and applied to find genetic polymorphisms in a wide variety of animals (Lessa 1992;Palumbi and Baker 1994;Corte-Real et al 1994;Daguin et al 2001;Chow and Takeyama 2000;Chow et al 2007). Primers designed to target conserved areas may have a wide utility across a broad range of animal taxa (Chow and Hazama 1998;Hassan et al 2002;Chow and Nakadate 2004;Pinho et al 2010;Jennings and Etter 2011). The universality of the primers for EPIC PCR depends on the conservation of exonic sequences, and the efficiency of PCR amplification may be primarily affected by gene copy number, including pseudogenes.…”

Regal phylogeography: Range-wide survey of the marine angelfish Pygoplites diacanthus reveals evolutionary partitions between the Red Sea, Indian Ocean, and Pacific Ocean

“…We resolved 431-bp of RAG2 using modified primers from 168 Lovejoy (1999); the forward primer is 5'-SACCTTGTGCTGCAAAGAGA-3' and reverse 169 primer is 5'-AGTGGATCCCCTTBTCATCCAGA-3'. We resolved 510-bp of S7 using primers 170 S7RPEX1F and S7RPEX2R from Chow and Hazama (1998). For each intron, PCR was 171 performed using the same reaction as described for cyt b but using the following temperature 172 conditions: 5 min at 94°C, 35 cycles of denaturing for 30 s at 94°C, annealing for 30 s at 58°C, 173 extension for 45 s at 72°C, and a final extension for 10 min at 72°C.…”

Polyphyletic Origin of Ornamental Goldfish

“…For amplification of cytb gene sequences of mtDNA primer pair FishcytB-F and THR-Fish-R was used [10]. For sequencing of the nDNA genes was used the following primer pairs: for rps7: S7RPEX1F and S7RPEX2R [11], for GH1 gene Cypr-GH1-1F 5'-AAAATGATTAACGACTTTG-3' and Cypr-GH1-2R 5'-CAAGTAGAAGTCCTCAAAA-3', for rhodopsin 545 Rh and Rh1039r [12]. mM each primer; 10 ng DNA, and 1 unit of Taq DNA polymerase.…”