PCR amplification of cDNAs of fish hemoglobin β chains using a consensus primer: cDNA-derived amino acid sequences of β chains from the catfishParasilurus asotus and the scadDecapterus maruadsi
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Hemoglobinβ chains were isolated from the catfishParasilurus asotus, the scadDecapterus maruadsi, the filefishThamnaconus modestus, and the scorpaenoidSebastiscus marmoratus by reverse-phase chromatography, and the N-terminal sequences were determined. To obtain the complete amino acid sequence, a 20-meric redundant consensus primer based on the N-terminal amino acid sequences of theβ chains was designed. Using this primer and oligo-dT adaptor, we amplified successfully the β-chain cDNAs of about 600 bp from the four fishes. The amplified products fromParasilurus andDecapterus were subcloned in theSmaI site of pUC18 and cDNA-derived amino acid sequences of 147 residues were determined, of which 69 and 76 residues, respectively, were identified by the chemical amino acid sequencing of internal peptides. Thus this PCR methodology using the consensus primer should be widely applicable for amplifying hemoglobinβ chains from teleosts.
Key wordsHemoglobin amino acid sequence catfish (Parasilurus asotus) scad (Decapterus maruadsi)
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