What are the differences between one-step and two-step RT--PCR?

Traditional two-step RT-PCR methods perform the reverse transcription (RT) reaction and the polymerase chain reaction (PCR) sequentially in separate tubes with separate buffers optimized for each step. One-step RT-PCR methods combine both the RT reaction and the PCR. A single buffer is optimized for both reactions, and the reactions occur in the same tube. The key benefits of a one-step approach are that they are more time-efficient, and reduce the risk of contamination by extraneous DNA. The disadvantage of one-step methods is that the fidelity of both the reverse transcriptase and the DNA polymerase enzymes can be reduced because the common buffer optimized to use the two enzymes in the same tube can actually be suboptimal compared to the buffers specifically optimized for each individual enzyme.

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