title: Degenerate oligonucleotide sequence-directed cross-species PCR cloning of the BCP 54/ALDH 3 cDNA: priming from inverted repeats and formation of tandem primer arrays.
abstract: Bovine corneal protein 54 (BCP 54) is the major soluble protein of the bovine cornea, and immunoreactive forms of this protein have been described in a wide range of mammals. Dideoxy sequence determination of a previously synthesized 420-bp cDNA to BCP 54 generated by the novel mixed oligonucleotide primer amplification of cDNA (MOPAC) procedure revealed extensive similarity to the cDNA encoding tumor-associated rat liver (class 3) aldehyde dehydrogenase (RATALD). PCR amplification with additional pairs of degenerate oligonucleotide sequence (DOS) primers derived from both BCP 54-amino-acid sequence and amino acid and nucleotide sequence data from RATALD produced three PCR products that were cloned and subsequently sequenced. The major product was 716-bp BCP 54 cDNA clone encompassing the BCP 54 carboxy-terminal amino acid sequence for which the DOS pair was designed. Sequence alignment of the BCP 54 cDNA and its translation product with RATALD demonstrated 81% and 85% identity at the nucleotide and amino acid levels, respectively. Analysis of the additional two clones established that they were the results of PCR artifactual processes. The first of these was a 552-bp product occurring at elevated primer concentrations that formed through bidirectional amplification from a single DOS annealing to an inverted repeat located in the BCP 54 coding sequence. The second artifactual product was a 212-bp sequence that contained several unreported amplification anomalies, including the formation of a tandem primer array.
authors.affiliation: Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710.
authors.forename: D L
authors.initials: DL
authors.lastname: Cooper
authors.forename: E W
authors.initials: EW
authors.lastname: Baptist
mesh_terms: D000444:Aldehyde Dehydrogenase / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D005136:Eye Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D051381:Rats; D012091:Repetitive Sequences, Nucleic Acid; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity
chemical_list: D005136:Eye Proteins; D015345:Oligonucleotide Probes; C030863:corneal protein 54, bovine; D004247:DNA; D000444:Aldehyde Dehydrogenase