Reverse Transcription question - ssproduct or dsproduct (Feb/10/2006 )

As far as reverse transcriptase is concerned, is there a difference in a first strand reverse transcription reaction and a double stranded reaction...when taking the cDNA product into Real Time reactions?

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Yes there is, when you start with single stranded you techincally lose one cycle (in the PCR) because the first cycle is just making the double stranded template then subsequent cycles are logrithmic in that both strands are copied... when you start with double stranded template all the cycles amplify logrithmically because both primers can bind for all the cycles...

HTH

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Second-strand synthesis involves the addition of T4 DNA polymerase with an incubation period, I believe, if you want all the template to be double-stranded.

This is necessary for in vitro transcription/RNA amplification etc.

-Matt

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Second-strand synthesis reactions generally also include DNA ligase (often an NAD-dependent ligase like E.coli DNA ligase rather than T4 ligase) to close nicks in the second strand if it has been polymerised from randomer priming. This is only an issue if you need a complete ds DNA for cloning - for RT-PCR most people do the second-strand synthesis during the first PCR extension phase, and tolerate the loss of one PCR amplification cycle. This is a pretty good reason to use an in-vitro transcript for calibration rather than a plasmid, if you're hoping for accurate absolute copy number estimation.

Del.

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