by Scott Cooper (University of WisconsinóLa Crosse, cooper@mail.uwlax.edu)

What is a primer?

A primer is a short synthetic oligonucleotide which is used in many molecular techniques from PCR to DNA sequencing.  These primers are designed to have a sequence which is the reverse complement of a region of template or target DNA to which we wish the primer to anneal.
 

Analysis of primer sequences

When designing primers for PCR, sequencing or mutagenesis it is often necessary to make predictions about these primers, for example melting temperature (Tm) and propensity to form dimers with itself or other primers in the reaction.  The following program will perform these calculations on any primer sequence or pair.

http://www.idtdna.com/html/analysis/Calculator.html

Some thoughts on designing primers.

1.  primers should be 17-28 bases in length;
2.  base composition should be 50-60% (G+C);
3.  primers should end (3') in a G or C, or CG or GC: this prevents "breathing" of ends and increases efficiency of priming;
4.  Tms between 55-80^oC are preferred;
5.  3'-ends of primers should not be complementary (ie. base pair), as otherwise primer dimers will be synthesised preferentially to any other product;
6.  primer self-complementarity (ability to form 2^o structures such as hairpins) should be avoided;
7.  runs of three or more Cs or Gs at the 3'-ends of primers may promote mispriming at G or C-rich sequences (because of stability of annealing), and should be avoided.

       (adapted from Innis and Gelfand,1991)
 

Practice Exercises