Searching a genome to find primer sequences that do not occur
Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Tue Feb 27 04:09:51 EST 2001
In article <3A9A87F3.D7613492 at gpu.srv.ualberta.ca>, the eminent Warren
Gallin at University of Alberta wrote
>Most sequences will not be there (4 to the 20th power possible
>sequences vs. the length of the actual genome you are looking at), so I
>don't understand what you are after. Do you mean take a list of
>sequences and see which ones are not in the genome?
MIcroarray's are produced by PCRing up a fragment of each individual
ORF. It appears to be fairly standard to put separate 5' and 3' common
tag sequences (14-15mer) on each ORF primer pair such that after the
initial PCR all purified ORF products can be re-amplified just by using
only one tag sequence pair as primers. This is not normally a problem
unless your tag primers happen to primer internally to your ORF, at
which stage it becomes a big problem.
I have just been presented with such a problem, not of our making, and
am wondering if there was an easier way to come up with a good tag
sequence. Rather than just picking a sequence at random and blasting
that against the genome I was wondering if there was a way of finding
sequences that don't exist. Unique tags could then be used based on
whatever criteria is then for selection i.e. GC clamp, %GC etc. etc.
It was just a thought especially as the tag pair I have been presented
with have virtually perfect match in some now problematic ORF's. They
can't have been blasted in the first place. My potential customer is
trying to blame PCR enzyme (not mine) rather than, as I have found,
their tag design! It is only a few ORF's with this problem but it is
still a problem.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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