Does RNA - PCR ??

Tracy Aquilla aquilla at salus.med.uvm.edu
Wed Jul 26 16:57:41 EST 1995


In Article <3uuo92$fcd at newsbf02.news.aol.com>, sehuang at aol.com (SEHuang) wrote:
>
>
>Several people in reponses  to the problem of PCR on RNA mentioned a RT
>activity of thermostable DNA pol. I think it is rather contamination with
>DNA that gives rise to the products. Even after rigorous DNAse treatment
>and digestion with 4-cutters restriction enzymes one might be able to
>amplify DNA in PCR. I had an artificial gene with an artificial
>mini-intron that allowed me to clearly distinguish between DNA (slightly
>longer amplicon due to intron) and RNA, and it was the DNA that is
>amplified. The band occured in the same strength in the No-RT control. (Or
>was it unspliced RNA ???)
>Nevertheless I am highly  interested in knowing more about the RT activity
>of Taq pol. I still cannot understand how the RNA:DNA hybrid generated
>after the first round of PCR by Taq pol should denature at 94 degree C (ie
>denaturation step in PCR ). RNA:DNA hybrids are more stable than DNA:DNA 
>duplex and usually do not separate at 90 dgree as I remember.  Does the
>Taq also has an RNAse H activity, like conventional reverse Transcriptases
>??? 
>Has anyone literarture about that ?
>Thank you for any suggestions, comments.
>
>Sui Huang
>Children's Hospital
>Boston

A few skeptics have responded with similar posts, so here are some references.

Anal Biochem 190: 292-6  (1990) [91150820]
Amplification, detection, and automated sequencing of gibbon interleukin-2 mRNA
by Thermus aquaticus DNA polymerase reverse transcription and polymerase chain
reaction.
A. L. Shaffer, W. Wojnar & W. Nelson

Gene 88: 293-6  (1990) [90269623]
Reverse transcription and direct amplification of cellular RNA transcripts by
Taq polymerase.
W. T. Tse & B. G. Forget

Methods Enzymol 218: 413-9  (1993) [93287861]
Reverse transcription of mRNA by Thermus aquaticus DNA polymerase followed by
polymerase chain reaction amplification.
M. D. Jones

Nucleic Acids Res 17: 8387-8  (1989) [90045979]
Reverse transcription of mRNA by Thermus aquaticus DNA polymerase.
M. D. Jones & N. S. Foulkes

Biotechniques 16: 282-4, 286-8  (1994) [94235290]
A coupled one-step reverse transcription PCR procedure for generation of full-
length open reading frames.
J. T. Aatsinki, J. T. Lakkakorpi, E. M. Pietila & H. J. Rajaniemi

RNA can be amplified by PCR. This is nothing new. FWIW, most of the time the
unexpected bands probably are from contaminating DNA, but not always.
    tracy



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