Time to Instigate Nested PCR

How to Obtain a Purer PCR Product and Reduce Non-specific Amplification

Unless you’ve gotten your hands on some miraculously specific primers, amplification of only your target sequence without non-specific amplification can be very challenging. Thankfully, a clever and surprisingly simple solution is at hand!

A Quick Recap of the Basics

In PCR, you design your primers to bind to the sequence you want amplified. A pair of primers is used, called the forward and reverse primers, to ensure both strands of your sequence are amplified. The hope is that after 30-40 cycles, you’ll have an almost pure product with which to work. Unfortunately, this hope is more like a dream when your primers are less picky than you’d like when it comes to what sequences they’ll bind. The end result is an impure product that may be unusable, depending on how much non-specific binding occurred and for what you need the PCR product.

A Simple Solution: Nested PCR

Nested PCR is a truly elegant solution. It uses two pairs of primers: the first set bind your target sequence but rather than binding closely to the beginning of the sequence, you design them to bind a little further away (by set we mean a forward and reverse primer). You run your PCR and end up with a product that contains both the target sequence and non-specific sequences. You then use a second set of primers, which have been designed as you would normally design primers, where they bind at or near the beginning of the target sequence.

Having two pairs of primers acts like a double check. There may be several sequences within your starting material that contains a string of bases to which one set of primers will bind but it is statistically very unlikely that they will contain base strings capable of binding both set of primers. Therefore, you can rest assured that your product is what you need it to be because of this extra level of selectivity. You can learn more about nested primers and their role in TAIL-PCR here and see a visual representation of the process here.

Additional Tips to Increase Your specificity

The specificity of PCR is determined by the specificity of the PCR primers. This is one of the cardinal rules of PCR. To increase the specificity of your primers, try the following:

1. BLAST Search
   Perform a BLAST search to see if your gene/sequence of interest has been sequenced before. If so, this could allow you to design highly specific primers.
2. Electronic PCR
   Another tech solution is Electronic PCR which allows you to run a computer simulation of the PCR process to check the theoretical outcome of your PCR.
3. Longer primers are less likely to bind other sequences since there is less chance of them binding efficiently enough to allow for amplification, although there is a balance and primers can be too long (see here.)
4. Too many G and C bases in a primer make it too sticky. Unfortunately, too few G’s and C’s and your primers are unlikely to anneal anything. There is a balance, but usually a GC content of 40—60% is ideal.
5. Find the optimal annealing temperature. This is the temperature at which at least 50% of the primers will bind their complementary sequence. If the temperature is too low, the primers become more likely to bind non-specific sequences. If you don’t know the optimal temperature, you can either use touchdown PCR (assuming you don’t need to know what the temperature turned out to be) or use a different annealing temperature for each well and see which product is the purest by gel electrophoresis (assuming you’d like to know what the optimal annealing temperature is).
6. An imbalance in the concentration of dNTPs can also cause problems, as can too high a concentration of dNTPs. Double-check your recipe with a colleague just in case.
7. Some additives can be used to destabilize DNA duplexes if you suspect that primer dimers or secondary structures like hairpins are causing a problem. First, try decreasing your concentration of magnesium chloride ions as this can reduce non-specific binding since higher concentrations of the ion stabilizes duplexes. Failing that adding DMSO may help. You can find a list of various additives for PCR here.
8. Failing all this, try a hot-start polymerase. These polymerases only work at higher temperature preventing amplification at lower temperature when the primers bind more easily to non-target sequences.

Please do add your own tips in the comments below.

References:

Thermal Asymmetric Interlaced PCR (TAIL-PCR) (to learn about one of the forms of PCR that relies on nested primers)

Nested Primers for PCR (for a visual on how nested primers work)

Liu, Y., & Chen, Y. (2007). High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences. BioTechniques, 43(5), 649–656

(to learn about how to increase the efficiency of your primers if you’re using TAIL-PCR)