cDNA synthesis and linear amplification!

Carrie carrie at fuzzyduck.freeserve.co.uk
Sun Feb 13 07:30:49 EST 2000


The article you are talking about is:

Sooknanan R and LT Malek (1995) NASBA. A detection and amplification system
uniquely suited for RNA. Biotechnology 13: 563 - 564.

Carrie

Harold E Smith <hes at U.Arizona.EDU> wrote:

> 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.

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?

Duncan wrote:

> > 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.







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