PCR problems

PCR Problems? Try an Additive

You’ve tried all the usual stuff, and checked the primer sequences twice, but still can’t get that PCR fragment amplified. It’s time to enter the strange world of PCR additives. Over the years a variety of additives have been shown to enhance PCR reactions in certain situations. Here is a summary of some of the PCR additives that have been described to help with PCR problems:

  • DMSO: Thought to reduce secondary structure that could inhibit the progress of the polymerase. Especially useful for GC rich templates. Use at a final concentration of 5-10%.
  • Glycerol: Similarly reduces secondary structure. Use 5-10%
  • Betaine monohydrate: Also acts on secondary structure formation. Use a final concentration of 1 to 3M.
  • BSA: Very useful for templates that may be contaminated with humic acids (e.g. environmental samples contaminated with soil) and is also reported to prevent reaction components from sticking to the tube wall. Use up to 0.8 mg/ml.
  • Tween-20: Can neutralize SDS left over from template DNA preparation that would inhibit the reaction. Use 0.25 to 1% final concentration.
  • Formamide: increases the stringency of primer annealing, resulting in less non-specific priming and increased amplification efficiency. Concentration range: 1-10%.
  • Tetramethyl ammonium chloride: Similar action to formamide. Try out concentrations from 10-100mM.
  • 7-deaza-2′-deoxyguanosine: A dGTP analogue that is especially useful for extremely GC rich templates. Success is reported with up to 83% GC. Use a 1:3 ratio of dGTP:7-deaza-2′-deoxyguanosine.

Read further for a more in depth discussion about some of these additives.  Or, If these don’t work for you, you can try some non-traditional additives.

The downside of PCR additives is that success or failure is very case specific – something that works with one PCR reaction will not necessarily work next time. Nevertheless, these reagents add another dimension to your PCR toolbox and although it is a very empirical approach, performing a few reactions with different additives can often get results, and dig you out of a frustrating situation.

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Originally published on September 2, 2007.  Updated and revised on April 29, 2016.

3 Comments

  1. mike on March 27, 2008 at 5:11 pm

    What literature is available for 7-deaza-2?-deoxyguanosine as an additive, and where can this be purchased?

    Thanks
    Mike

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