Primers are essential for initiating DNA amplification, whether for the purposes of detection, cloning or sequencing. The specificity of PCR requires that the primers specifically bind to the target DNA and not to other non-target DNA. The primer you design impacts the entire DNA amplification process. Thus, it is crucial to understand how to successfully design primers. Some tips for efficient primer design are as followings.
Tips for PCR primer design
1. Primer Length: The optimal length for PCR primer is generally 15-30 bases. This length is long enough for adequate specificity and short enough for primers to bind easily to the template at the annealing.
2. GC Content: The GC content of primers is generally 40%-60% with the 3’ end of a primer in G or C to promote specific binding due to stronger hydrogen bonding of G and C bases and help with the stability of the primer. More than 3 G's or C's should be avoided in the last 5 bases at the 3' end of the primer as this can cause primer-dimer formation.
3. Primer Melting Temperature (Tm)
: Primers with melting temperatures in the range of 52-58 °C within 5°C of each other generally produce the best results. Primers with melting temperatures above 65°C have a tendency for secondary annealing. In order to verify the Tm, try an annealing temperature gradient PCR reaction to find the optimal Tm according to your primer and enzyme.
There are many ways to calculate the Tm value, such as according to the formula Tm=4(G+C) +2(A+T).
4. Restriction Enzyme Site: Where a restriction site has been added onto the end of a primer, typically, 5-6 nucleotides are added 5’ of the restriction enzyme site (aka a “leader sequence”) in the primer to allow for efficient cutting.
5. Three Bases: Try to avoid runs of 4 or more of one base, or dinucleotide repeats (for example, ACCCC or ATATATAT) as this can cause primer mispriming.
6. Avoid Template Secondary Structure: Avoid regions of secondary structure; namely intra-primer homology (more than 3 bases that complement within the primer) or inter-primer homology (forward and reverse primers having complementary sequences). These circumstances can lead to self-dimers/hairpins or primer-dimers instead of annealing to the desired DNA sequences. Thus have a balanced distribution of GC-rich and AT-rich domains. Toolslike IDT Oligo Analyzer can help you to detect secondary structure.
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