On Fri, Mar 23, 2012 at 12:54 PM, Nathan McCorkle <nmz787@gmail.com> wrote:
> if that's the case, don't you want to do an alignment of all the
> possible sequences for your gene, the few highest scoring alignments
> would seem to be the primer you want. (are you doing PCR, or is this
> for hybridization/blot assays?)
>
We do PCR, but we know the mutation site. To build the primers, we go
through a sequence(size can be a few hundred bp long) around our PCR
site of interest. We expect to find a place where the sequences are
highly identical to our query. Sometimes, we find a base mutation that
is common in all our hits so we can "wobble" that base. We like to
make the primer in a "clean" location, i.e., there is not a lot of
variation in the mutations along the possible primer site.
I attached a file I worked on. We got 6000 hits for this 60bp BLAST
query. We looked through a total of 500bp to find a good primer site.
You can see in the file that the 3rd bp has Thymine as a common base,
so we can wobble that. Eventually, we determine the best site to build
a primer by analyzing all the Hits together.
It is very time consuming, and I want to automate at least half of the
process. All this sorting and editing with MS WORD and EXCEL is
time-consuming and tiring on my eyes. The boss is used to it and he
doesn't mind.
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