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.