I'm sure people are getting sick of my postings so let me begin by apologising.
My latest problem is as follows. I'm studying a part of a family of genes
that are remarkably similar. High homology at the DNA level as well
as multiple transcripts due to alternative splicing resulting in small
differences in some cases and larger ones in others.
I have to deal with 6 possible transcripts at the moment and I have the
cDNA sequences of all of them. In order to identify which transcript(s)
are ppresent in the cell types I study I'd like to use PCR primers
to generate sequences unique to each transcript.
So far, I've been designing primers using PRIMER from the Whitehead
Institute and it does a workmanlike job most of the time in allowing
you to specify thermodynamic and nucelotide requirements.
However when you delimit a target sequence and regions of interest this
imposes constraints on Primers ability to design good primers so I get
none even when I relax the stringency or primer design.
*So what is really needed is a primer design program that can look at*
*a multiple sequence alignment and then try and look for primers*
*that will define sequences unique to each of the elements in the*
*set of aligned sequences as well as allowing you to define parameters*
*like GC content and Tm.*
As far as I know such a package does not exist. What's the alternative?
It seems to me that there must be a better way to design these primers
than taking them one strand at a time and finding them to be promiscuous
with respect to the others or relaxing 3' complementarity, Tm, GC content
All comments and suggestions are welcomed.