PCR and reverse dot hybridization for the detection of endogenous retroviral transcripts.
Two degenerated oligonucleotide primers, known to amplify a fragment of the pol gene in all retroviruses tested so far have been used to amplify pol related sequences from human genomic DNA. Cloning and sequencing these fragments confirm a retroviral relationship for most of them and define 96 groups on the basis of their internal similarity. 96 pol fragments were probed with PCR amplified cDNA in reverse dot hybridization to investigate pol related transcripts. PCR amplified genomic DNA served as a control for contamination of genomic DNA in the RNA preparations. Isopycnic centrifugation in cesium trifluoroacetate yielded RNA with the lowest possible amounts of contaminating DNA. This technique is a powerful and a well-controlled tool for the detection of endogenous retroviral transcripts and may be helpful for investigating the involvement of endogenous retroviruses in various diseases.
Herrmann
M
M
Department of Medicine III, Friedrich-Alexander-University Erlangen-Nürnberg, Germany.
Kalden
J R
JR
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D015751:Genes, pol / Q000235:genetics; D015894:Genome, Human; D006801:Humans; D016086:Lentivirus / Q000201:enzymology / Q000235:genetics; D008180:Lupus Erythematosus, Systemic / Q000235:genetics; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000201:enzymology / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D012194:RNA-Directed DNA Polymerase / Q000235:genetics*; D012190:Retroviridae / Q000201:enzymology / Q000235:genetics*; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid
D017931:DNA Primers; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase