In search of retrotransposons: exploring the potential of the PCR.
A rapid and universal procedure for isolating reverse transcriptase encoding elements from diverse mammalian genomes using PCR is described. We have designed short, degenerate primers to conserved amino acid domains of retroviral reverse transcriptase. These primers amplify a region, predicted to be 342-396 base pairs for most mammalian retroviruses, that spans several conserved domains of reverse transcriptase. The region encoded by the amplified PCR product contains a number of highly conserved amino acids that aid in identification of either degenerate reverse transcriptase or reverse transcriptase from new, undescribed elements. Additionally, these primers allow the amplification of a piece of DNA large enough to be used for phylogenetic analysis. The primers have been used successfully to isolate a region of three related reverse transcriptases from two mammalian taxa. The generality of this approach is discussed.
Wichman
H A
HA
Department of Biological Sciences, University of Idaho, Moscow 83843.
Van Den Bussche
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004251:DNA Transposable Elements; D006801:Humans; D008322:Mammals / Q000235:genetics / Q000382:microbiology; D008969:Molecular Sequence Data; D010542:Peromyscus / Q000235:genetics / Q000382:microbiology; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011533:Proviruses / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification*; D012194:RNA-Directed DNA Polymerase / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D012190:Retroviridae / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification*
D004251:DNA Transposable Elements; D012194:RNA-Directed DNA Polymerase