We are trying to design primers for SNPs that have little to no flanking region on the 5' end. Are there any suggestions for the most efficient way to do this? Is the best option to PCR with a single primer and then sequence? Will there be enough product to sequence in this linear PCR? Thanks in advance for any tips!
Cycle sequencing in fact relies on linear amplification rather than conventional bi-directional amplification. So, if the primer is unique, i.e. it has single binding site in the genome, one is expected to get clean peaks in sequencing chromatograms. This is the way exactly how large insertions in genome are sequenced by walking.
you do not say how you generate these sequences but is there enough material to blunt end clone then sequence using plasmid based primers ? Why cannot the sequences be found in one of the many genome databases sequenced online ?
We're working with a non-model organism that has no published genome.
You might need to find out the sequence right outside your allele's 5'-end by some available molecular methods, such as iPCR. Then, use this new-found sequence to design a 5' end primer.
There are actually 2 SNPs in this fragment, one with ~250bp after and one with ~10bp after.
you might consider artificially created restriction sites ACRS ( also published as DCAPS which googles to a site that designs your primer )
essentially almost any sequence can be changed into a restriction site by the deliberate changing of a close by base so that the combination of the polymorphic base and the introduced base can be forced to ampilfy by lowering the annealing temperature then cut to reveal the sequence when resolved on an agarose gel. Since you have so few bases on the end position you would need to add a tail to the acrs primer to make it easy to view the cut product size and in some cases to add sufficient bases to allow cutting to take place and also ensure that the forward primer is very good quality preferably unique as you will lose much specificity with only 8 or 9 true bases at the polymorphism end of the sequence. It is generally a good technique particularly for screening large numbers of samples
I agree with Paul, we have used this method successful in regions where the reference sequence contained NNNNN after the SNP. Good luck!
How large is the genome, in kilo- or mega-bases? If it is a microorganism with a genome 15MB or less, there is a technique for sequencing directly off the genome with one primer. It requires highly pure DNA, like that made with a Qiagen kit. I developed a method for yeast, and it worked well to identify transposon insertion sites. Because the transposons were inserted randomly, the only information I had was the transposon sequence, which I used as the sequencing primer. A description of the method and protocol for yeast is in: Horecka J, Jigami Y. 2000. Identifying tagged transposon insertion sites in yeast by direct genomic sequencing. Yeast, 16: 967–970. It has also been applied in bacteria (smaller genome, see references in my paper). Good luck!
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