I think we all have been through those my-PCR-product-didn’t-get-amplified days. Sometimes, playing around a bit more with the PCR-conditions brings luck, or sometimes it doesn’t work at all. These days we have access to many different types of DNA polymerases, ultrapure and buffered nucleoside triphosphates, and other necessary starting materials in convenient concentrations; but still we have to take care of the two critical components of a PCR reaction: nucleic acid template and the oligonucleotide primers.
Selection of the right set of functional primers plays an important role in the overall success of the PCR reaction. How should you do it?
1. Short or long enough: Primer length
The more nucleotides you add, the more specific the primer becomes; but then, the shorter the primer, the more quickly it anneals to the template DNA forming a stable, double-stranded DNA polymerase binding site. Balancing this dilemma, 18-24 bases are thought to be very sequence specific and work efficiently.
You can also use primers which are shorter or longer depending on what you want to amplify. For e.g use primers as short as 15 bases when mapping simple genomes and 28-35 bases when you want to amplify a sequence with high degree of heterogeneity.
2. Temperature is important: Tm and Ta
A melting temperature (Tm) range of 56°- 62°C is important for efficient annealing and also provides a sufficient thermal window to optimize from. Avoid using primers having Tm higher than 65°C because they tend to result in secondary annealing.
You can estimate the Tm if your primers are shorter than 20 bases using the simple and roughly accurate calculation: Tm=2°C × (A+T) + 4°C × (G+C) or you can use an online tool to calculate the Tm value of a given primer set.
So we have talked about the Tm of a single primer alone, does it have anything to do with the Tm of the other?
A big yes because you need an annealing temperature (Ta) that is compatible with both! Avoid using primers with Tms that vary greater than 2°-5°C from each other because the primer with the higher Tm will have a higher chance of mispriming at the lower temperature while the primer with the lower Tm might not be functional at a very high temperature.
You can calculate the best fitting Ta value online as well. But sometimes, you have to try out different annealing temperatures in order to get the right product, why not try a gradient PCR?
3. High or low bondage: GC content
The Tm of the primer is determined by the type of bases: higher numbers of G and C increase the Tm. Try to keep the GC content in the range of 30-60% for optimum amplification. Don’t forget to match the GC content and the Tm within a primer pair. Also try to avoid having a GC clamp (more than 3-4 G’s or C’s consecutively) within your primer sequences.
4. Ends matter: 3’ and 5’ end of the primer
The 3’ end of a primer is extremely critical for a successful PCR; especially the last 5-6 nucleotides. Minimal mismatch and perfect base pairing between the 3’ end of the primer and the target DNA template is important. In addition, if you can, include a few G’s or C’s in the last few bases of the 3’ end. These bases will bind to the template strongly, thus increasing the specificity.
The 5’ end of the primer is less critical. You can add unrelated sequences at this end, for e.g. when you need to insert a restriction site for cloning. Remember to calculate the accurate Tm for these primers containing 5’ overhangs: only use the sequence specific portion of the primer for a good result.
5. Repeat and secondary formation of primer
Try to avoid long stretches or repeated di-nucleotides stretches in your primer. If you have a long stretch of di-nucleotides, for e.g. ATATATATA it can result in mispriming. In addition, repeated patterns can result in primers that form hairpin loops or self-dimers, or the primer pair may even be complementary and bind together. You can use online tools to check these parameters as well.
6. Product length
The length of the PCR product also contributes toward the overall efficiency of the amplification. More than 3kb long products can be amplified from pure plasmid or high molecular weight DNA template. If you want to detect a specific DNA fragment, for e.g. in a clinical assay, choose a length of 120-300 bp. You can select a product length of 250-750bp while monitoring a gene expression by qPCR.
7. Cross binding: check for homology
After you have taken care of all of the above, check the unique specificity of the primer; you want make sure the primers will not bind anywhere else apart from the targeted region.
8. Computer software or yourself
Designing and selecting the correct oligonucleotide primer pair can be tedious. Fortunately, there are computer programs that make it easier. You can specify different parameters such as Tm, GC content, product length etc. depending on your requirements. The software program will then choose primer sets that best suit your experiment. A word of caution: make sure you understand the function of the software before you start using it.
So spend a little more time to design a high quality primer set before ordering and starting your experiment. You still may have to optimize the PCR to get it to work efficiently. But, trust me, it is worth the smile you will have after a successful PCR reaction.
Dieffenbach C.W. , Lowe T.M.J, and Dveksler G.S. 1993. General Concepts for PCR Primer Design. Genome Research. 3: S30-S37