DMSO - is it useful in the Reverse Transcription reaction

Ashok Aiyar axa12 at po.CWRU.Edu
Fri Jan 31 23:45:10 EST 1992


In a previous article, srounsle at sdcc3.ucsd.edu (Steven Rounsley) says:

>I have been watching the discussion of the use of DMSO or formamide
>in PCR reactions to break down secondary structure.  Does anyone
>have any experience of using it to disrupt secondary structure of
>RNA during a reverse transcription reaction, prior to amplifying a
>desired sequence from the resulting cDNA.  You see my problem may
>be that my desired sequence isn't even making it as far as the cDNA
>because of this secondary structure, so using DMSO in the PCR will
>not help.

Use AMV reverse transcriptase at 42 - 45 degrees.  Additionally, drop
the KCl concentration to below 15 mM (final reaction concentration).
Adding DMSO has little positive effect on viral reverse transcriptases.

You may want to try Perkin Elmer's recombinant Tth polymerase which has
RT activity in the presence of Mn as cation.

Ashok
-- 
  Ashok A. Aiyar   Department of Biochemistry   CWRU Med. School
   		       axa12 at po.cwru.edu
		   aiyar at cwbio.bioc.cwru.edu
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