competitive RT-PCR

helene dauchel helene.dauchel at univ-rouen.fr
Fri Mar 13 06:24:03 EST 1998


I have the project  to use competitive RT-PCR. To construct the
competitor I ask me if RNA competitor during the RT step is realy
necessery or if DNA competitor added just during the PCR step can also
be a good approach?

In the case of a RNA competitor, a poly A+ tail must be present to be
used during the RT step with an oligo (dT). I have seen two
possibilites to generate these poly A+ tail: 
-first: during the generation of the DNA competitor by using a
composite primers including an oligo (dA) and then clonage and
transcription in vitro in a vector type pBS.
-second: after transcription in vitro using a transcriptional vector
which have both a RNAse polymerase promoter and a poly A+ sequence. Do
these kind of vector exist??

Who can tell me which are the best solution  ?


Thank's for any help

helene






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