How to Amplify Difficult PCR Substrates

During my postgraduate studies, I did literally one PCR reaction with a pre-optimised protocol on a not especially difficult template. So my karma came back with vengeance, when as a part of my first postdoc I had to amplify a template containing a 35 bp-long GC-rich stem-loop, which proved to be extremely difficult. This was my first encounter with difficult templates, but not the last. After deconstructing PCR one step at a time, I came up with the following strategies for amplifying difficult templates.

Template

Make sure that you have enough good quality template DNA. Trust me, it’s all too easy to blindly add not enough and/or dirty template. So it’s worth your  time to run an aliquot of your template DNA on a gel and analyse a sample in a spectrophotometer, which shows contamination level – especially if you got your samples from somebody else. Even if you are sure that your DNA is not degraded, you may want to clean it up or make a new sample – it does wonders for any PCR reaction, including difficult PCRs.

Annealing

1. If you are sure that your problems are not caused by DNA quality or a dodgy dNTP mix, make sure you know why your template is difficult, e.g. high GC or AT content, strong secondary structures or a long template – 10 kb is too much for an ordinary Taq polymerase. For the first two cases, you can try using  additives. DMSO had helped me to amplify 35 bp stem-loop, and I know that other people in my lab have used it successfully. BSA doesn’t hurt but sometimes helps. You may also consider using one of these additives (one at a time, please). An XXL template may require the use of special polymerases, for example Phusion. 2. Make sure that your primers are optimal. If possible, start with a couple of C or G residues (ideally at both ends, but the 5’ end is more important) – this increases the annealing strength. The primers should not contain strong secondary structures and, if the sequence allows it, should have a GC content of approximately 50-60%. The minimum primer length is 20 bp; increasing it by 10 bp increases annealing as well. Primer pairs should have annealing temperatures within 5°C of each other. Sometimes moving your primer 20 – 30 bp upstream or downstream will help.

Extension

3. You can generally increase the likelihood of amplifying your target by decreasing the stringency of your PCR reaction. This is achieved by:

• lowering the annealing temperature to 45 – 50°C;
• increasing the magnesium concentration, which improves amplification (at least of AT-rich substrates). Standard buffers for PCR reactions usually contain 2 mM Mg; this can be increased up to 10 mM.
• The extension time is usually about 1min/per kb of template, but it can be doubled. Let the machine work while you sleep.

4. You may want to try “touch–down” or “touch-up” PCR protocols. These protocols start from a high (“touch-down”) or low (“touch-up”) annealing temperature for several cycles, then increase the temperature by a couple of degrees for several cycles, etc. The idea behind these protocols is that at some point, your primers anneal (at their optimum temperature) and start amplification.

Amplification

5. Increase the number of cycles from 30 to 40, and try adding more enzyme after 20 cycles. 6. Using a gradient PCR machine, which is capable of checking multiple annealing temperatures at a time, saves you a lot of work. If you combine it with different Mg concentration and/or polymerases, you will cover a lot of bases in one go (pun intended). If the DIY approach doesn’t help, you may consider using special mixes and polymerases optimised specifically for difficult templates, for example DyNAzymeEXTDNAPolymerase or FastStart–Taq. I would be happy to hear about your tips for amplifying difficult templates, specifically, which commercial products work in which cases, because for some unknown reason I trust colleagues more than the ads.