Reverse transcriptase fidelity

Mansky LM, Temin HM (1995) Lower in vivo mutation rate of human immunodeficiency virus type 1 than that predicted from the fidelity of purified reverse transcriptase, J Virol 69 5087-5094... 

A limitation of this strategy is the fidelity of enzymes—reverse transcriptase and DNA polymerase— used in PCR to produce consistently error-free DNA sequences. These enzymes typically introduce error at the rate of one base per 400... 

Pandey VN, Kaushik N, Rege N, Sarafianos SG, Yadav NS, Modak MJ. Role of M184 of human immunodeficiency virus type-1 reverse transcriptase in the polymerase function and fidelity of DNA synthesis. Biochem 1996 35 2168-2179. 

Wainberg MA, Drosopoulos WC, Salomon H, Hsu M, Borkow G, Parniak MA, et al. Enhanced fidelity of -selected mutant HIV-1 reverse transcriptase. Science 1996 271 1282-1285. 

The human immunodeficiency virus type 1 (HIV-1) belongs to the family of positive-stranded, enveloped RNA viruses with a DNA intermediate step (retroviruses). Because of the lack of fidelity of the reverse transcriptase (RT), the replication is error-prone, and the infection is characterized by its quasispecies nature. Antiretroviral treatment with such compounds as zidovudine (AZT), zalcitabine (ddC), didanosine (ddl), stavudine (d4T), and lamivudine (3TC) select for quasispecies variants that are resistant to these compounds (1). The detection of these variants is clinically important because they may affect the outcome of the treatment (2). 

After internalization, the virus is uncoated in preparation for replication. The genetic material of HIV is positive-sense singlestrand RNA (ssRNA) the virus must transcribe this RNA into DNA to optimally replicate in human cells (transcription normally occurs from DNA to RNA—HIV works backward, hence the name retrovirus). To do so, HIV is equipped with a unique enzyme, RNA-dependent DNA polymerase (reverse transcriptase). Reverse transcriptase first synthesizes a complementary strand of DNA using the viral RNA as a template. The RNA portion of this DNA-RNA hybrid is then partially removed by ribonuclease H (RNase H), allowing reverse transcriptase to complete the synthesis of a double-stranded DNA (dsDNA) molecule. Unfortunately, the fidelity of reverse transcriptase is poor. 

Choi, J.Y. and Guengerich, F.P. (2004) Analysis of the effect of bulk at N2-alkylguanine DNA adducts on catalytic efficiency and fidelity of the processive DNA polymerases bacteriophage T7 exonuclease- and HIV-1 reverse transcriptase. 

Alternatively, 0.5 pi (4-5 U) AMV reverse transcriptase from Promega (Cat No. M5101) and 0.5 pi (2 U) of Taq DNA polymerase from Boehringer Mannheim (Expand high fidelity PCR system, Cat No. 1732641) can be mixed and added to the reaction mixture. 

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