Can Taq use RNA as a template?

tfitzwater at NEXSTAR.COM tfitzwater at NEXSTAR.COM
Thu Sep 9 19:30:35 EST 1999


M. D. Jones and N. S. Foulkes, 1989.  Reverse Transcription of mRNA by
Thermus aquaticus DNA polymerase.  Nucleic Acids Res. 17(20):  8387-8388.
The authors report that RT activity by Taq DNA polymerase has an optimum Mg
requirement of 2-3 mM (the dNTP concentration was not defined).

During the SELEX process, we PCR amplify with 1 mM @ dNTPs and 7.5 mM
MgCl2.  There appears to be RT activity under these conditions.  Although
the evidence is rather compelling, I am not completely convinced that, in
our case, contamination is not a problem.  All of our RNA, oligos and
primers are gel-purified (PCR products are not gel-purified). I have
evidence that nucleic acid from a previous purification can survive rather
extensive gel plate cleaning operations and transfer to the acrylamide of a
subsequent gel.  This contaminant is then eluted with the desired gel band.


Tim Fitzwater
NeXstar Pharmaceuticals (Gilead Sciences)

>From: greg.appleyard at usask.ca (Greg Appleyard)
>Subject: Can Taq use RNA as a template?
>Date: Thu, 09 Sep 99 14:04:02 EST

>Can Taq act as a DNA polymerase using RNA as a template?
>We are curious about the origins of amplified fragments following PCR in
>our RNA+ but cDNA and DNA negative control lane.

>Greg Appleyard
>Western College of Veterinary Medicine




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