Reverse transcriptase inhibits Taq polymerase activity.
Detection of viral RNA by polymerase chain reaction (PCR) requires the prior reverse transcription of the viral RNA. In order to minimise the number of manual manipulations required for processing large numbers of samples, we attempted to design a system whereby all the reagents required for both reverse transcription and amplification can be added to one tube and a single, non-interrupted thermal cycling program performed. Whilst attempting to set up such a one-tube system with Taq polymerase (Taq; Biotech International) and avian myoblastosis virus (AMV) reverse transcriptase (RT), we noticed a substantial decrease in the sensitivity of detection of viral RNA. Investigation of this phenomenon has revealed direct interference of RT with Taq polymerase. Evidence supporting this conclusion includes the following observations: (1) increasing the ratio of Taq to RT improves sensitivity; (2) adding non-homologous RNA improves sensitivity; (3) RT that has been heat inactivated prior to Taq addition does not exert this effect; (4) the effect is not sequence restricted; (5) the Mg2+ ions are not sequestered by RT. In addition, the effect is not limited to AMV RT, Moloney murine leukaemia virus RT also affects Taq activity.
Sellner
L N
LN
Department of Microbiology, University of Western Australia, Nedlands.
Coelen
R J
RJ
Mackenzie
J S
JS
D009189:Avian Myeloblastosis Virus / Q000201:enzymology*; D001483:Base Sequence; D005784:Gene Amplification; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000201:enzymology*; D019384:Nucleic Acid Synthesis Inhibitors; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D012680:Sensitivity and Specificity; D019914:Taq Polymerase; D013696:Temperature
D019384:Nucleic Acid Synthesis Inhibitors; D012367:RNA, Viral; D019914:Taq Polymerase; D012194:RNA-Directed DNA Polymerase; D008274:Magnesium