Does Taq have reverse transcriptase activity? - (Aug/31/2005 )
I've perform a reverse transcription control without reverse transcriptase to see if my sample contains genomic DNA. I previously digested RNA with DNase I RNase free and I made sure of completness of DNA digestion. Then I perform PCR of b-actin and I got amplification even in the RT minus control. So I think that if Taq has got reverse transcriptase activity then RNA template for reverse trancription is being amplificated in the subsequently PCR. what do you think?
Umm I don't think Taq pol (at least pure one) has reverse transcriptase activity. Are you sure you didn't use a combined RT-PCR enzyme mix?
And how are you sure that your DNA was completely digested? I'd say the evidence in your RT neg control shows that it wasn't.
I think you have contamination buddy or else your DNaseI Tx didn't work well; or else you have made a million dollar discovery!
If Taq did have RT activity then the -RT negative control wouldn't be informative. Listen to the fellas posting above me - double check for DNA/sample contamination.
First thank you all for your attention.
When I first saw amplification in RT minus control I thougth that DNase I didn't worked well. So I took isolated genomic DNA and I perform several digestion with increasing amount of DNase I. Then I saw that the concentration of DNase I that I used to get rid of the contaminant genomic DNA in my RNA sample totally digested 2 ug of genomic DNA (that is much more than the amount of contamination). That's why I can say that there's virtually no genomic DNA in my RNA sample after treatment with DNase I.
Well, my PCR kit is: Hot start Taq DNA polymerase from Quiagen. so If you think we could be in front of a important evidence feel free to investigate (of course I would aprecciate if you give me credit in your paper )
To confirm that RNA was amplificated I am going to digest minus RT control with RNase A. By the way, I have liofilizated RNase, do you know the apropiate buffer to disolve it?
Thanks again fellows
No, it does not. You must have genomic DNA contamination. You are going to have to purify again.
This is why it is advantageous to work with sequences containing introns. The cDNA and genomic DNA appear as separate bands.
May be your others PCR reagent have contaminated with DNA. What about the water that you used?
actually DNA polymerase can have RT activity in the right conditions
Myers, T.W. and Gelfand, D.H. (1991): Reverse transcription and DNA
amplification by a Thermus thermophilus DNA polymerase. Biochemistry, 30, 7661-7666
but this will not happen in the lab
more likely is that you have DNA contamination in your Taq
I forgot to mention that I also performed a control without template in which there was no amplification. So neither the water or any other component was contaminated with genomic DNA.
The primers that I used to amplify b-actin are desing against two different exons. So as it was mention, I was able to differentiate amplification between a genomic sequence and a messenger sequence. But you must keep in mind that sometimes genomic DNA contains the sequence of a gene without introns (pseudogene). That could be my case because when I amplify b-actin from purified genomic DNA I got the amplicon without introns.
actin has at least 40 pseudogenes
you are probably seeing amplification of one of these
try and amplify something intronic from your sample and isee f you still get a positive result