PCR primers anneal to template DNA to initiate DNA replication by DNA polymerases. The strength of annealing, as measured by the primer melting temperature, has been shown to be critical for PCR amplification. In addition, PCR primers should have minimum self-annealing and minimum random annealing to non-target sequences.

Guidelines for selecting PCR primer sequences are as follows:

• 1. Forward and reverse primers should have similar melting temperatures.
• 2. Primers should not form self-annealing primer-primer dimers. The following primers form primer-primer dimers, so shouldn't be selected.

            P1: 5'-...GGCGATCG-3'
                        ||||||
                     3'-GCTAGCGG...-5' : P1 
  or 
            P1: 5'....AGGGCCC-3'
                         || |
                      3'-CGCGAAT...-5' : P2 
  

• 3. A balanced AT/GC content is preferred.
• 4. G or C at the 3'-end of a primer is preferred.

PCR annealing temperature is usually chosen to be 4-6 degree below the primer melting temperature. There are different formulas for calculating melting temperature. Melting temperature is defined as the temperature at which 50% of primers are in the bound state. The most widely used formula is based on the number of AT/CG counts:

The nearest neighbor method (Jhon SantaLucia, Proc. Natl. Acad. Sci. Vol. 95, p1460-1465 (1998) ) is more accurate, but difficult to calculate. Fortunately there are now several online primer design programs that save users from these time-consuming calculations:

• Primo (Chang Bioscience, http://www.changbioscience.com/primo/) is a friendly online program for primer design. Users can chose from a variety of options, including the choices of melting temperature formulas. Primo also has an option to eliminate primer candidates that match over-represented transcribed sequences, therefore reduce background random primering in RT-PCR reactions.
• Web Primer (Stanford, http://genome-www2.stanford.edu/cgi-bin/SGD/web-primer/) designs both PCR and sequencing primers.