cDNA synthesis and linear amplification!
Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Mon Feb 7 08:16:05 EST 2000
In article <389e9989 at redeye.it.ki.se>, Csaba Kiss <csakis at ki.se> writes
>I am wondering if anybody tried to amplify the amount of mRNA during the
>first strand cDNA synthesis. The idea is very simple. You use oligo(dT) for
>priming, do the synthesis, then heat the sample to 80-85 degree, (maybe 94)
>and let the sample cool down and add reverse transcriptase again. This way
>you double the amount of mRNA you had in the beginning. If you do it again,
>you triple your mRNA. In some application you need very high amount of
>polyA+ RNA, then this would solve the problem, and you do not introduce PCR
>generated bias or artefacts.
>Did anybody try this method?
The odd possible snag. RNAseH activity of the RT may destroy the
template if you are using wt AMV or wt MMuLV Rt. Maybe the RNaseH- MMuLV
RT would work. The high temp denaturation in the presence of Mg may also
destroy the RT. Finally if the RNA is intact, will it anneal
preferentially to the cDNA rather than the primers.
Will it work? I'll let you all know.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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