5 prime Race-where do you start?

Clive Tregaskes Clive.tregaskes at bbsrc.ac.uk
Wed Oct 9 15:29:36 EST 1996


Hi everyone, here's a little problem that you might be able to help me
with. I'm trying a 5' RACE (using dC tailing of 1st strand cDNA) to
find the transcription start site of the gene I'm studying.  PCR seems
to work OK, out of 10 clones picked, 5 contain part of the transcript.
Four of these all end around the same point (but not exactly!!) and
the last one extends the possible mRNA approx 400 bases beyond this.
Either
1 two start points (tho' Northern suggests this isn't so)
2 short transcripts are due to secondary structure stopping the RT
3 long transcript is due to DNA contamination of my RNA prep (but
contains a dC tail)
Lots of ideas but no answers, so does anybody know how you tell the
real start site from any secondary structure artifacts.
Thanks in advance for your help
Clive Tregaskes
Inst. Animal Health
Compton
Newbury
UK

Clive.tregaskes at bbsrc.ac.uk




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