DMSO - is it useful in the Reverse Transcription reaction

[Steven Rounsley]

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.

If anyone has any experience of this (positive or negative) I would
be interested to know.

Thanks,

Steve Rounsley

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Steve Rounsley : " The comments made here reflect the opinion
of nobody"
srou...@ucsd.edu

[Ashok Aiyar]

In a previous article, srou...@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
ax...@po.cwru.edu
ai...@cwbio.bioc.cwru.edu
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