cDNA synthesis and linear amplification!
Harold E Smith
hes at U.Arizona.EDU
Fri Feb 11 14:55:23 EST 2000
I recall a recent (within the past six months) article for linear
amplification of mRNA. The first strand synthesis used an oligo dT primer
with a T7 promoter at the end. The finished cDNA was used as template for
T7 RNA polymerase amplification, which could then be used for a second
round of cDNA synthesis. If you'd like, I'll track down the reference.
On Mon, 7 Feb 2000, Dr. Duncan Clark wrote:
> 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....
> Duncan Clark
> DNAmp Ltd.
> Tel: +44(0)1252376288
> FAX: +44(0)8701640382
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