Minimizing DNA recombination during long RT-PCR.

Recent developments have made it possible to reverse transcribe RNA and amplify cDNA molecules of > 10 kb in length, including the HIV-1 genome. To use long reverse transcription combined with polymerase chain reaction (RT-PCR) to best advantage, it is necessary to determine the frequency of recombination during the combined procedure and then take steps to reduce it. We investigated the requirements for minimizing DNA recombination during long RT-PCR of HIV-1 by experimenting with three different aspects of the procedure: conditions for RT, conditions for PCR, and the molar ratios of different templates. We used two distinct HIV-1 strains as templates and strain-specific probes to detect recombination. The data showed that strategies aimed at completing DNA strand synthesis and the addition of proofreading function to the PCR were most effective in reducing recombination during the combined procedure. This study demonstrated that by adjusting reaction conditions, the recombination frequency during RT-PCR can be controlled and greatly reduced.