Polymerase chain reaction, an automated process for DNA replication, is most accurate 80 to 150 bases away from the primer site. As a result, primers should be designed based on the sequence of DNA that is 80 to 150 nucleotides, or bases, away from the site of interest.
Primers are most effective when they are 18 to 30 nucleotides long, and preferably 20 to 25 nucleotides long. The melting temperature should be between 131 and 167 degrees Fahrenheit. The nucleotide composition should be 40 to 60 percent G-C, guanine and cytosine. For best results, use a computer program for this step of the primer design.
A primer will anneal, or bind, to sequences that are complementary to the primer. Because we want the primer to anneal to the DNA sequence of interest, it is important to avoid primers that are complementary to themselves. If primers are self-complementary they will anneal to other copies of the primer rather than to the DNA strand.