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multiple products in primer extension

David L. Haviland, Ph.D. HAVILAND at KIDS.WUSTL.EDU
Mon Jul 25 12:49:17 EST 1994


In <Ct8s98.7z1 at sci.kun.nl> koend at sci.kun.nl writes:

> Dear netters,
> 
> I am currently trying to map the tsp of the Plasmodium falciparum pfs25
> gene, which is expressed in a stage-specific manner. The A/T content of
> the untranslated region is about 90 percent.
> When I perform a primer extension according to Current Protocols or Maniatis,
> either using AMV-RT/Superscript RT or rTth RT, I see a large number of extension products. The pattern is never the same.
> I have made an in vitro transcript that mimics the putative in vivo transcript.
> When I perform a primer extension on this transcript I observe, as expected, one single product. When I perform a primer extension on Plasmodium falciparum RNA lacking the pfs25 transcript, I observe no product at all. 
> Apparently, the RNA containing the pfs25 transcript contains material (a-specific sequences/inhibitors?) that blocks a proper extension, or gives rise to a-specific products.
> The primer I use is a 25-mer with a Tm of approx. 55 C. The RNA I use is isolated using a RNAzol reagent and looks beautiful on a Northern blot.
> Does anyone has suggestions on how to solve this problem? I would be extremely grateful if you would share them with me. Please feel free to contact me directly by e-mail. I will repost a compilation of all suggestions.
> Koen Dechering

Koen:

Some random thoughts:  Having done this procedure from the same book, I've 
learned a few things along the way.  I would consider upping your oligo to 
at LEAST a 40-mer.  I think the problem is that with a Tm of 55'C, along 
with using 50% formamide you are not going to get consistient hybridization 
of your oligo to your RNA.  I'd be willing to bet that if you calculated 
your Tm based on the Tm=16.6Log[M] + 0.41[Pgc] + 81.5 - Pm - B/L - 
0.65[Pf], where M is molar conc of Na+, Pgc is percent G/C bases, Pm is 
percent mis-match, Pf is percent *formamide* (note it is a negative 
number), B is 675 for synthetic probes up to 100 bp, and L is the length of 
the probe in bases... that you might find your actual Tm is probably down 
in the 30's range: MUCH too low for accurate hybridization;thus, you get 
many non-specific sites because of 1) primer annealing and 2) RNA folding.

In addition to a new and longer oligo, I would suggest punching through 
this formula and make your Tm no lower than 50'C and adjust the %formamide 
accordingly.  As this has worked for me, I hope it will do so for you...

Hope this helps,
David
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