PCR amplification - basic question

[esl]

Hello,

I am currently trying to teach PCR to another member of our lab - who asks a lot of questions and has revealed my own lack of understanding. Please help!

After first-strand cDNA synthesis, and once the first short strand product is generated, does the first (long) strand remain in the reaction? And does it contribute significantly to the final product? What about the mRNA strands - are these degraded and therefore not primed in the PCR?

Thanks so much - we are getting really confused trying to understand properly.

[jonoave]

Interesting question; and as far as I know:

1. The first-strand should remain in the reaction, though I do not see the significance. Isn't the first-strand and second-strand complementary to each other? Ie. amplification of the second-strand would produce another copy of the first strand, and so on...

2. Whether the mRNA remains depend on the steps you took during the reverse-transcription process. Eg. in Invitrogen's First-strand synthesis, in the last few steps, you add in RNAse H. Which I believed is used to degrade the mRNA. On the other hand, for Ambion, there is no such step.

I don't believe the presence of mRNA would impact the PCR later, what is more important is the RT enzyme. Generally, in reverse transcription, one of the last few steps involve incubating the reaction at high temperatures to deactivate the RT enzyme. This is to prevent the RT enzyme activity during subsequent PCR reactions. Therefore, even if you have some remaining mRNA in the cDNA sample, it should not be an interference when the RT enzyme has been deactivated.

[Aga]

The second question was answered above
To the answer for the first question I can add that first strands of the template remain in the reaction (theoretically intact) and are amplified in one direction (untill the temperature changes, since only one primer binds to these strands). After first 4 - 6 cycles the amplification of these long strands has little contribution to the overal reaction efficiency, so there's nothing to worry about.

The presence of long initial template strands usually don't influence the final product. There isn't any possibility to amplify long fragments of these strands because temperature is changed rapidly during the reaction.