I am sure many of you have been there. Everything is going smoothly, and your project seems to be working out perfectly. And then there is this one PCR. For some reason, it just won’t work. It is a black dot on your record. Even though I have a scientific mind, I have to be honest, I sometimes believe in witches. Well, scientific witches—by which I mean: valid, solid reasons, for why a PCR is not working! It is so frustrating to spend time and energy on a PCR, and then go to check your agarose gel full of enthusiasm to find no band, a lousy smear, or many unspecific bands. In this article I will give you some answers that may help you to solve all your problems. Or at least, your PCR problems.
First Things First… Look at Your Template
Take a good, hard look at your template. Get to know as much as possible about it. Does it have high GC content (over 65%)? If it does, beware. A high GC content will probably make your template much harder to amplify, but don’t despair, you can address this. To improve amplification, you may increase the annealing temperature, and/or add DMSO or add another secondary structure destabilizer to ensure that your GC rich template will be amplified.Primer Design
Primer design is extremely important. Here are just some of the tips to keep in mind while designing PCR primers:- Primer length: Primers should be around 18–22 nucleotides in length. This way, they are long enough to ensure specificity (by decreasing the probability of binding non-target sequences), yet short enough to bind to the template.
- Secondary structures: secondary structures will keep your primers busy among themselves, instead of just annealing to your template. Luckily, there are some bioinformatic tools that can predict the likelihood of secondary structure formation in primers, such as OligoAnalyzer 3.1.
- Melting and annealing temperature: Melting temperature (Tm) is calculated using the AT:GC content of your primers and is provided to you by your primer supplier. For your PCR to work, the double stranded primers need to be single stranded in order to anneal to your template—this is accomplished by heating your reaction to the proper Tm. Primers that have Tms in the range of 52–58 ºC generally produce the best results. Annealing temperature is the temperature at which the single stranded primers will anneal to the template: if the annealing temperature is too high the annealing process won’t be successful leading to low PCR product. However, if it is too low, then non-specific hybridization may occur, which leads to non-specific PCR products.
- Specificity: This is super important. If your primers are not specific, you can never be certain of the identity of your PCR product. Make sure your primer will only align with your target, and not any other sequence (e.g., pseudogenes) in your DNA template! You should always blast your primer sequence against your reference genome sequence before ordering any new primers to minimize problems with specificity.
- It is also a good idea to make sure your primers don’t anneal to any known polymorphism/mutation. Remember that allele dropout can happen if a primer pair can´t anneal to one allele, and this can lead to the loss of precious information. It sounds scary, but there are many bioinformatic tools that will help you accomplish the perfect primers.