Hey what is the accurate procedure for insilico-primer design for STR loci?

Question

[gskbioinfo143]

Hey i want to design insilico primers for CODIS 13 STR loci (CSF1PO, D3S1358,D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, THO1, TPOX, vWA)

i need suggestion whether to take SNP sequence of loci or Sequence from refseq..

Hope to hear from u

Thanks

Answer 1

[Digiomics]

I am not sure what you mean with 'SNP sequence'.

Do you need to design your own primers? There are already plenty of published primer sequences for each of the STR loci, see here for example http://www.cstl.nist.gov/strbase/coreSTRs.htm.

If you do need to design your own, I would base the design on the human genome sequence. By that you can benefit from mapped data, such as known SNPs.

I would start by determine the exact genome coordinates of the repetitive region for each locus, for example by blasting the reference sequence given in the STRbase against the genome sequence. Keep in mind that the reference sequence probably represents a different allele than the human genome sequence, so do not except perfect matches.

I would then extract the STR sequences plus some additional bases up- and downstream to design primers. In order to get robust primers, I would then check if the designed primers contain any known SNP sites. This can be done via the genome coordinates, see here for example: http://www.ncbi.nlm.nih.gov/variation/view/?cfg=NCID_1_20164020_130.14.22.10_9146_1426149575_1381684626

Additionally, I would check if my primers are specific by using an ePCR tool.