Minimizing DNA recombination during long RT-PCR

Recent developments have made it possible to reverse transcribe RNA and amp lify cDNA molecules of > 10 kb in length, including the HIV-1 genome. To us e long reverse transcription combined with polymerase chain reaction (RT-PC R) to best advantage, it is necessary to determine the frequency of recombi nation during the combined procedure and then take steps to reduce it. We i nvestigated the requirements for minimizing DNA recombination during long R T-PCR of HIV-l by experimenting with three different aspects of the procedu re: conditions for RT, conditions for PCR, and the molar ratios of differen t templates. We used two distinct HIV-I strains as templates and strain-spe cific probes to detect recombination. The data showed that strategies aimed at completing DNA strand synthesis and the addition of proofreading functi on to the PCR were most effective in reducing recombination during the comb ined procedure. This study demonstrated that by adjusting reaction conditio ns, the recombination frequency during RT-PCR can be controlled and greatly reduced. (C) 1998 Elsevier Science B.V. All rights reserved.