Unknown DNA — Rational Primer Design and Analysis — the ...

Features” window for one of my downstream primers is illustrated below against a backdrop of the Editor
window using a “16:1” zoom ratio so that the entire sequence will fit in the window at once:
Test for Specificity —Will Your Primers Only ‘Find’ the Correct Genes? GCG’s FindPatterns
Another valuable test of any candidate primers should be done before running off to or sending off your
request to the oligo’ synthesis lab. You should check for your primers’ specificity to insure that your primers
will not hybridize to completely the wrong type of sequence by checking them against the DNA database.
This step can also point out, and allow you to correct if necessary, errors in your primer sequence created in
the backtranslation step, if enough DNA sequences are available in the database to allow a comparison. The
GCG program FindPatterns is probably best for this. It can be used to screen your candidate primers against
the entire DNA database, any of the subset databases, or any other sequences desired. There are several
advantages to using FindPatterns over standard similarity searching software: 1) you can test more than one
primer at a time against as many sequences as you want; 2) the algorithm will not allow any gapping of your
primers to the template, which would represent loop structures in the hybrid and should not be allowed; 3)
similarities don’t count — identities are required but mismatches are allowed by option; again, just what you
want in primer analysis; and 4) word size parameters are not relevant since the algorithm doesn’t use them,
therefore, they don’t have to be messed with (which you would need to do if you were using heuristic style
similarity searches such as BLAST since they are not designed to find short regions of DNA similarity). For
these reasons, I do not recommend using BLAST or FastA style searches for testing primer specificity. The
easiest way to run FindPatterns is to provide it with your primers as an input file rather than typing them in
interactively. To do this FindPatterns needs its input list of patterns to be in exactly the same pattern data
format as described above for Prime. This makes it relatively easy to test them against the database.
However, running a full-blown FindPatterns GenBank search would require too much time for the time
constraints of our lab session. If you would like to run this type of analysis for your own research, it is very
32