Reverse transcriptase inhibits Taq polymerase activity

Abstract

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.

Cloning and characterization of a complete open reading frame of the hepatitis C virus genome in only two cDNA fragments.

“…Previous studies have shown that the activity of Taq polymerase is reduced by an active reverse transcriptase (Sellner et al, 1992). It was recommended therefore to inactivate the reverse transcriptase, e.g.…”

The Role of the CC Chemokine, RANTES, in Acute Lung Allograft Rejection

“…This format allowed optimal amplification with little or no nonspecific amplification of contaminating DNA, as determined by the absence of bands when RT was excluded. After amplification, the sample (20 l) was separated on a 2% agarose gel containing 0.3 g/ml (0.003%) of ethidium bromide, and bands were visualized and photographed using a UV transilluminator (33)(34)(35).…”

Helicobacter‐induced expression of Bcl‐XL in B lymphocytes in the mouse model: A possible step in the development of gastric mucosa‐associated lymphoid tissue (MALT) lymphoma

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