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Primer Design

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.
 

imageDFM

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.

The programs will calculate both the Tm of the primers, as well as any undesireable pairings of primers.  When primers form hairpin loops or dimers less primer is available for the desired reaction.  For example...
 
Hairpin

Dimer

Some thoughts on designing primers.

Also keep in mind that most oligonucleotide synthesis reactions are only 98% efficient.  This means that each time a base is added, only 98% of the oligos will receive the base.  This is not often critical with shorter oligos, but as length increases, so does the probability that a primer will be missing a base.  This is very important in mutagenesis or cloning reactions.  Purification by HPLC or PAGE is recommended in some cases.
 
 
Oligonucleotide length
Percent with correct sequence
10 bases
(0.98)10 = 81.7%
20 bases
(0.98)20 = 66.7%
30 bases
(0.98)30 = 54.6%
40 bases
(0.98)40 = 44.6%

Practice Exercises

Primer Design 1

Primer Design 2