PCR Primer Design
PCR copies a stretch of DNA between two short single-stranded primers: a forward primer that matches the start of the region and a reverse primer that matches the reverse complement of its end. Good primers bind only where you want them, at about the same temperature, without sticking to themselves.
Forward and reverse primers
The forward primer has the same sequence as the start of the top strand, so it pairs with the bottom strand and is extended to the right. The reverse primer is the reverse complement of the end of the region, so it pairs with the top strand and is extended to the left. Both are written 5'→3', the direction the polymerase builds DNA.
Example: for a region that starts ATGGCTAAGCTT and ends GGTCCATAGTAA, the forward primer is ATGGCTAAGCTT and the reverse primer is the reverse complement of the end, TTACTATGGACC.
The key properties
Length trades specificity (longer is more specific) against binding efficiency (shorter binds more easily). GC content keeps binding stable without making the primer sticky. The Tm must suit the cycling temperatures, and the two primers should match; see melting temperature. A GC clamp, one or two G/C bases in the last five at the 3' end, helps the polymerase start, but too many make the end bind in the wrong place.
Primer dimers and hairpins
A hairpin forms when part of a primer is complementary to another part of itself and folds back. A self-dimer forms when two copies of the same primer pair up; a cross-dimer when the forward and reverse primers pair. Dimers that include the 3' ends are the worst: the polymerase extends them and makes a short junk product (primer-dimer) that competes with your target. Repeats and runs such as GGGG or ATATAT let a primer slip and bind at the wrong place.
Specificity
Even a perfect-looking primer can bind other places in a genome. Check candidate pairs against the genome or transcriptome with a tool such as NCBI Primer-BLAST before ordering.
Primers for qPCR, cloning and mutagenesis
qPCR works best with short products, about 70 to 200 bp, and primers of similar Tm; ordinary end-point PCR tolerates products from a few hundred bases up to about a kilobase.
Cloning primers carry a restriction site on the 5' end. Add a few extra bases in front of the site, because most enzymes cut poorly at the very end of a DNA fragment. Design the part that binds the template first and add the tail afterwards, so the tail does not change the way you judge Tm.
Site-directed mutagenesis primers are longer, often 25 to 45 bases, with the change near the middle and 10 to 15 matching bases on each side.
How Aaron's Archive does it
| Part | What the tool does |
|---|---|
| Single sequence tab | A quick starting suggestion: the first 20 bases as the forward primer and the reverse complement of the last 20 as the reverse primer, each scored for GC content, Tm and 3' end. |
| Primers tab | Takes a template of at least 60 bp (up to 1,000,000) and tries forward primers that start within the first 80 bases and reverse primers that end within the last 120, so the product covers most of the sequence and is at least 100 bp. Each pair is scored for GC content, Tm and 3' end; hairpins and self-dimers cost 2 points per primer, a cross-dimer costs 3, and a Tm difference of 2 °C or less earns a bonus. The best-scoring pairs are listed with their flags. Tm is nearest-neighbor. |
| Not checked | Whether a primer also binds elsewhere in a genome. Check that separately. |
Before you order
Treat any computed primer as a first draft. Confirm Tm with the calculator your supplier uses, check specificity, and be ready to run an annealing-temperature gradient.
Common questions about primer design
How long should a PCR primer be?
Usually 18 to 25 bases. Shorter primers bind less specifically; longer ones bind more slowly and are more likely to form dimers.
What GC content should a primer have?
40 to 60%, and ideally avoid long runs of the same base. See GC content.
What is a GC clamp?
One or two G or C bases at the 3' end of a primer. They bind more firmly where the polymerase starts, but more than three G or C in the last five bases can make the primer stick in the wrong place.
What Tm should primers have?
Roughly 55 to 65 °C, with the forward and reverse primers within 2 to 3 °C of each other. See melting temperature.
What causes primer dimers?
Two primers (or two copies of one primer) whose 3' ends are complementary, so the polymerase extends them using each other as a template. They show up as a small band of about 30 to 60 bp on a gel.
Can I try this on my own sequence?
Use the PCR primer design tool: it returns eight ranked pairs with Tm, GC, hairpin and dimer checks.