erhugo@magnus.acs.ohio-state.edu (Eric R Hugo) (04/29/91)
I am soliciting opinions on an experimental method for pulling a cDNA out of RT mRNA. Perkin-Elmer/Cetus has recently developed a system that uses a novel DNA polymerase called (I think) rTth DNA polymerase. It is a thermostable enzyme that has the unique property of being a reverse transcriptase in the presence of Mn and at 70 C. In the presence of Mg at 60 C it acts like Taq polymerase. I have a degenerate primer made to the N terminal aa sequence of the enzyme that I'm studying (mixture of 64 seqs, 20 mer). I attempted to use this oligo to screen several cDNA lambda libraries. After over 1.5 million plaques screened I gave up. With only one primer PCR at the time did not seem to be the answer. What I am considering now is using rTth, my degenerate primer, and oligo dT as the other primer to try to amplify a cDNA from oligo dT selected RNA. I do not believe that my degenerate oligo is bad as I can get a signal with it on Northerns and Southerns. Does anyone out there have an opinion as to whether this sounds feasable or am I grasping at straws? Any opinion would be greatly appreciated. Please E-mail comments to: _______________________________________________________________________ | Eric R. Hugo email: Hugo.1@osu.edu | | Dept. Of Molecular Genetics ATTnet: (614) 292-6804 | | The Ohio State University :If it can't be fixed with | | 484 W. 12th Ave. :duct tape, | | Columbus, Ohio 43210 :it can't be fixed. | -----------------------------------------------------------------------