Specific suppression of false-positive signals in the product-enhanced reverse transcriptase assay.
The recently developed ultrasensitive reverse transcriptase (RT) test involving a PCR step can detect minute amounts of RT in single retroviral particles and is 10(6) times more sensitive than conventional RT assays. We have found that different DNA-dependent DNA polymerases like DNA Polymerase I from Escherichia coli and eukaryotic enzymes like DNA polymerase alpha and gamma exhibit RT-like activities in this assay, whereas DNA polymerase beta and Klenow enzyme show only minor activities. To discriminate false-positive signals caused by DNA-dependent DNA polymerases in the RT-PCR assay, we have included increasing amounts of activated DNA in the RT reactions. This modification of the assay leads to complete inhibition of aberrant but not authentic RT activities. This improvement specifies the RT-PCR assay as the most sensitive tool for the detection of even very rare replication-competent retroviruses and of related enzymatic activities indigenous, for example, for products of endogenous retrovirus-like RT genes in cell extracts.
Lugert
R
R
Paul-Ehrlich-Institut, Langen, Germany.
König
H
H
Kurth
R
R
Tönjes
R R
RR
D001483:Base Sequence; D017795:Bromovirus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004256:DNA Polymerase I / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000201:enzymology; D005189:False Positive Reactions; D009052:Leukemia Virus, Murine / Q000201:enzymology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012194:RNA-Directed DNA Polymerase / Q000032:analysis / Q000378:metabolism*; D012190:Retroviridae / Q000201:enzymology*
D017931:DNA Primers; D004247:DNA; D012194:RNA-Directed DNA Polymerase; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase