Designing Polymerase Chain Reaction Primers Using Primer3Plus

UMMS Affiliation

Program in Bioinformatics and Integrative Biology; Department of Biochemistry and Molecular Pharmacology

Publication Date


Document Type



Bioinformatics | Computational Biology


Designing oligonucleotide primers is a crucial step for successful molecular biology experiments that require the use of the polymerase chain reaction (PCR). PCR involves cycles of three steps: denaturation, annealing, and extension. During denaturation, double-stranded DNA (dsDNA) molecules (templates) are separated into single strands. During annealing, a pair of primers is annealed to the complementary regions of the single-stranded molecules. In the extension step, DNA polymerase extends the primers to produce DNA molecules that correspond to the region bracketed by the primers (the amplicons). All of these steps are temperature sensitive, and the common choice of temperatures is 94 degrees C, 60 degrees C, and 70 degrees C, respectively. Poorly designed primers may lead to no amplification product or additional undesired amplified fragments. The goals of primer design include good primer specificity, high annealing efficiency, appropriate melting temperature, proper GC content, and the prevention of primer hairpins or primer dimers.

DOI of Published Version



Cold Spring Harb Protoc. 2016 Sep 1;2016(9):pdb.prot093096. doi: 10.1101/pdb.prot093096. Link to article on publisher's site

Journal/Book/Conference Title

Cold Spring Harbor protocols

Related Resources

Link to Article in PubMed

PubMed ID