Designing a good pair of PCR primers is probably the single most important factor for successful PCR reactions. A primer is a short, synthetic, single-stranded DNA sequence that is complementary to the template region of DNA. PCR reactions require two complementary oligonucleotides primers; forward primer and reverse primer. Forward primer anneals to the region upstream of the DNA segment to be amplified and the reverse primer anneals to the region downstream of the DNA to be amplified.
Primers are the key to the success or failure of a PCR experiment. If the primers are designed correctly, the experiment results in the amplification of the target region of the template molecule but if the primers are incorrectly designed, the experiment will fail possibly because no amplification occurs or the wrong fragments get amplified. Designing a working primer can be a bit tricky, with so many ways to design them and a number of small details that must be taken into consideration.
The process of designing specific primers typically involves two stages.
- The primer’s flanking regions of interest are generated using bioinformatics tools.
- The primer’s pairs are searched against a nucleotide sequence using tools such as the basic local alignment search tool (BLAST) to examine the potential target.
PCR Primer Design Consideration
There are several properties you’ll need to consider while designing successful PCR primers. Some of them are;
- Primer specificity
- The length of the primer
- Annealing and melting temperatures of the primers
- The GC content of the primer,
- GC Clamp and the
- Secondary structure of the primer
Design primer that is complementary to the template region of the DNA. It must be ensured that the likelihood of annealing to sequences other than the gene of interest is very low. Primer specificity is usually dependent on the length and annealing temperature. A sequence homology search (eg, primer blast) with known template sequences in the public genome database like NCBI helps determine its specificity.
PCR primers of 18-24 base pairs are optimal to get the best yields. The length is long enough for adequate specificity. Also, it is short enough for primers to bind easily to the template at the annealing temperature.
If your primers are too short, they could produce more non-specific DNA amplification products. But on the other hand, if they’re too long they can result in slower hybridization rates.
Annealing and Melting Temperatures
The annealing temperature is the temperature at which the primer hybridizes with the template DNA. Annealing temperature affects the specificity of the reaction. If the annealing temperature is too high, the primers and the template remain separate but if the annealing temperature is too low, primers may bind nonspecifically to the template.
The ideal annealing temperature must be low enough to enable hybridization between the primer and the template DNA but high enough to prevent mismatch hybrids from forming. The annealing temperatures of both primers should be close to each other. Annealing temperature should be five degrees below the melting temperature so that most of the primers bind to the template. Lowering the temperature any further, however, could result in more non-specific binding.
The melting temperature (Tm) is a temperature where half of the primers will dissociate from the DNA. The two primers (forward primer and reverse primer) should ideally have similar melting temperatures. Primers with Tm in the range of 52-58°C generally produce the best results. Primers with melting temperatures above 65°C have a tendency for secondary annealing.
If the Tm of your primer is very low, including a sequence with more GC content, or extending the length of the primer a little may help.
The GC content of the sequence gives a fair indication of the primer Tm. There are many tools that can help to calculate Tm online but there’s an easy way you can quickly do the rough math. Simply add four degrees celsius for every G or C, and two degrees celsius for every A or T in your primer sequence. This calculation is based on Wallace-Itakura formula for calculating oligonucleotide melting temperatures;
Tm= 2 (A+T) + 4 (G+C)
GC content of the primer
Higher GC content (the number of G’s and C’s in the primer as a percentage of the total bases) ensures a more stable binding between the primers and the template DNA because G-C base pairs are stronger than A-T base pairs. The ideal GC content of a primer is 40-60%. For example, if a primer contains twenty base pairs total, and has six Gs and four Cs, it would have a 50 percent GC content.
GC base pairs are useful because they have three hydrogen bonds, one more than the AT pairs. Therefore, GC pairs help promote stronger and more specific binding to the template DNA.
You’ll also want to include a GC clamp at the 3′ end of the primers. Including two to three Gs and Cs to the 3′ ends of the primer helps promote specific binding between the primer and the template.
Primers should have two to three Gs and Cs at the 3′ end to bind more specifically to the template DNA but more than 3 G’s and C’s should be avoided as this can cause primer-dimer formation.
Depending on the sequence of the primer there’s a chance it could form hairpins or dimerize with itself or the other primer. This means it could base pair with itself or base pair with the other primer instead of the template.
Primer dimer (PD) consists of two primer molecules that have hybridized with each other instead of the template because of strings of complementary bases in the primers. To prevent primer dimerization, you should avoid the presence of identical nucleotides between forward and reverse primers (i.e. inter-primer homology).
Also, be aware of the presence of repeats in your nucleotide sequence. Too many repeats could cause mispriming, and the primer can anneal to unintended locations. To prevent dimerization and mispriming, avoid running 4 or more of one base, or dinucleotide repeats (for example, ACCCC or ATATATAT). Similarly, avoid intra-primer homology i.e., the presence of more than 3 bases that complement within the primer.
Various online tools can be used that warn potential hairpins, self-dimers, or cross-dimers formation in a particular nucleotide sequence. For example, MFEprimer-3.0 is a tool that can be used to check the quality of PCR primers. It is used for checking the presence of non-specific amplicons, dimers, hairpins, etc in the candidate primers.
If your primes do not give you the PCR product you’re looking for, you could try changing the condition of your PCR reaction like the annealing temperature. If still not having any luck, it might be time to go back to the drawing board and redesign your primers.
If you know the gene sequence of your gene of interest and want to amplify the whole gene, then designing a primer can be pretty straightforward.
- Forward primer: First 20 nucleotide sequences of your gene of interest can be the primer sequence for your forward primer.
- Reverse primer
- Select the last 20 nucleotide sequences of your gene of interest.
- Past that sequence code in the text box in this link.
- Press reverse complement
- The gene sequence that you get is your reverse primer.
The manual selection of optimal PCR primer sets can be quite tedious so currently, suitable PCR primers are chosen using software programs or online tools such as primer BLAST, eurofins primer design tool, etc. Prior set selection algorithms of primer designing software help to select the best primer candidate by calculating and analyzing different sequences as per parameters (such as melting point, GC content, primer length, etc) specified by the users.
Primer designing using NCBI primer BLAST
You can also design the PCR primers using NCBI primer BLAST tools. Check this link for the detailed methodology
Uses of Primers
Apart from amplifying the target gene of interest in PCR methods, primers are also used in DNA sequencing and other experimental processes such as genotyping (to detect sequence variations in alleles in specific cells or organisms), cloning DNA fragments of interest, and mutagenesis (introduction of desired mutations into the gene of interest to study gene expression and other attributes).
References and further reading
- Kun Wang, Haiwei Li, Yue Xu, Qianzhi Shao, Jianming Yi, Ruichao Wang, Wanshi Cai, Xingyi Hang, Chenggang Zhang, Haoyang Cai, Wubin Qu, MFEprimer-3.0: quality control for PCR primers, Nucleic Acids Research, Volume 47, Issue W1, 02 July 2019, Pages W610–W613, https://doi.org/10.1093/nar/gkz351.
- PCR Primer Design Tips. ThermoFisher Scientific. Retrieved on 24 June 2022.
- How to Design a Primer for PCR. Addgene. Retrieved on 24 June 2022.
- PCR Primer Design Guidelines. Premier Biosoft. Retrieved on 26 June 2022.