As you know PCR Reactions require two complementary oligonucleotide primers. Primers are short, single stranded complimentary DNA fragments that anneal to the regions upstream and downstream of the DNA segment to be amplified.
Designing a working primer can be a bit tricky, with so many ways to design them and a number of small details that can derail your reaction.
There are several properties you will need to consider to design succesful primers.
- The length of the primer.
- The anealing and melting temperature of the primers.
- The GC content of the primer.
- The secondary structure of the primer.
1) The length of the primer -
Generally you will want your primers to be between 18 to 24 base pairs. This is the optimal range to get the best yields. If your primers are too short they could produce more non-specific DNA amplification products. meaning that some of your DNA products are not what you were trying to amplify.But on the other hand if they are too long they can result in slower hybridization rates.
2) The anealing and melting temperature of the primers -
Your annealing temperature is what allows the primers to base pair with the DNA.The melting temperature is a temperature where half of the primers will dissociate from the DNA. You want the kneeling temperature to 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 two primers should ideally have similar melting temperatures. There are many tools that can help you calculate this online but there's an easy way you can quickly do the rough math for yourself.
Simply add four degrees celsius for every G or C, and two degrees celsius for every A or T in your primer sequence.
Great so we have the length of the primer and the annealing and melting temperatures set. But what about the makeup of the
3) Nucleotides in your primer -
The ideal makeup of a primer is between 40% and 60% GC content. The GC content is the percentage of Guanines and Cytosines in the primer.So for example, if a primer contains 20 base pairs total, and has 6 Gs and 4 Cs, It would have a 50 percent GC content.
You will also want to include a GC clamp at the 3' prime end of the primer. This means that you include two to three Guanines and Cytosines to the three prime end of the primer if possible, given the constraints of your DNA sequence. GC base pairs are useful because they because have three hydrogen bonds, one more than the AT pairs that have two hydrogen bonds. Therefore GC pairs help promote stronger stronger and more specific binding to the template DNA, helping to ensure your reactions will work properly.
4) The secondary structure of the primer -
Depending on the sequence of the primer there is a chance it could form hairpins or dimerize with itself or the other primer. This means it could base pair with it self or base pair with the other primer instead of the template.Too many repeats could cause mispriming, and the primer can anneal to unintended locations. Avoid runs of identical nucleotides in the three prime portion of your primer too, for the same reason.
You just successfully designed a PCR primer! If your primers do not give you the PCR product you're looking for You could try changing the conditions 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.
