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.
koend at sci.kun.nl