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I am trying to design primers for sanger sequencing snp detection. In the past I have used Primer-BLAST, but I'm trying to pick up KRAS codon 12 mutations. The problem is that in Primer-BLAST you specify refseq gene ID and so it doesn't seem to design primers outside of the gene locus. This means that my primers will sit on the SNP. Is there a way to tell Primer-BLAST the exact locus I am trying to sequence? Or is there a better way of doing this? Thanks.

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You can manually restrict the design ranges in Primer-BLAST screen.

enter image description here

  1. First of all you should enter the targeted sequence. In this case, it will be NC_000012.12 which corresponds to chromosome 12 that has KRAS gene.
  2. You should determine the position of variant that you interested in that chromosome. The KRAS G12D -or rs121913529- variant is in 25245350. position. You can find the position of specific variant on dbSNP.
  3. For Sanger sequencing, the recommended size is 600-1000 bp. So, we should select ranges +/- 300-500 bp apart from the variant position.
  4. For the left primer we choose From:25244850 To:25245050; for the right primer we choose From:25245650 To:25245850. And then submit.
  5. Here are the primer-sets that you are looking for. :)

enter image description here

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    $\begingroup$ So actually for the way I phrased the question, this is the correct answer and I appreciate it this works for me trying to get G12D. But the problem I am trying to get at as well is what if the variant I am trying to pick up is in one of the first 100 bases listed in the RefSeq accession number. So the for primer 'From' number should be something like -350. Can this be done? $\endgroup$
    – The Nightman
    Apr 27, 2015 at 20:43
  • $\begingroup$ I understand your question. Unfortunately, it is not possible. I suggest that instead of using RefSeq ID, you can enter larger sequence -such as chromosome-. $\endgroup$
    – Can H.
    Apr 27, 2015 at 20:50
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    $\begingroup$ yeah that makes sense, that's sort of what I figured. Thanks. $\endgroup$
    – The Nightman
    Apr 28, 2015 at 1:51
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For sequencing primers, I've always used this tool to great effect. You can set up how many bases you want to include before your target, and define how to divide large exons and set up no. of overlapping bases. I think this is what you ask for in your comment to the previous answer. http://ihg.gsf.de/ihg/ExonPrimer.html

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  • $\begingroup$ glad to be of service :) $\endgroup$
    – WesternBlöd
    Apr 29, 2015 at 21:22

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