Sequence Comparison
Align the sequences by species Look for single base errors, insertions and deletions
Choose a ‘type’ sequence for each species Compare potential SNPs with SNP data in the refseq file header
Align the ‘type’ sequence for each species Look for potential splice variants in one species not reported in the one of interest
Splice Variants
Primer & Probe Selection
Available Software for Designing Taqman® Assays Primer Express®, Applied Biosystems Primer3 from The Whitehead Institute @ MIT:http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi
Using Primer Express®, Personal Tricks 1- For sequences over 1 Kb, use a 500 bp subsequence 2- Choose the area based on a region with an even Tm content from the ‘Map’ tab
Primer & Probe Selection
Using Primer Express®, Personal Tricks
3- Under the ‘Rxn Cond’ tab, set the primer concentrations to 200 nM from 50 nM
4- Under the ‘Params’ tab, set the G/C clamp to 1 from 0
5- Keep the %G/C content of the primers and probe as close as possible during selection
6- Match the Tms of primers & probe as a set eg., 59°, 59°, 69°C
7- Keep the total number of G&Cs between 2-3 in the last 5 bases of the 3’ end of the primers
8- Probes should not start with a G
9- Avoid probes with more than 3-Gs in a row or those with much higher G than C content, make the reverse probe.
10- Select 3-4 primers around a probe to test
11- Check the sequence alignment to make sure the assay is in an acceptable region
