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Another blank gel after a failed PCR? Are you having problems amplifying GC-rich regions of DNA?
No, it isn’t you that’s the problem, and you’re certainly not alone if you’re having trouble amplifying GC-rich sequences or understanding why GC-rich sequences are causing such issues in the first place!
Problems amplifying GC-rich regions by PCR have been an irritant for scientists for decades!
There are several options available if you are having problems getting good amplification of GC-rich regions, which alone or in combination may help you deal with this problem.
But first, let’s look at why GC-rich sequences are more difficult to amplify.
What is Causing Your Problems with Amplifying GC-rich Regions?
Problem 1: Thermal and Structural Stability
Firstly, what do we mean by GC-rich regions? When we say “GC rich”, we mean approximately 60% of the bases are either cytosine (C) or guanine (G).
GC-rich DNA sequences are inherently more stable than sequences with a low GC content. For PCR, this means that the higher the GC content, the higher the melting point of the DNA.
The increased stability of GC-rich DNA sequences is, contrary to popular belief, not primarily because of the hydrogen bonds. Stabilization is mainly due to stacking interactions called base stacking. There are some beautiful biochemistry and biophysics behind why this stacking occurs. (1)
This is why Thermus thermophilus, an extremophile that needs to tolerate very high environmental temperatures, has a GC-rich genome. This is also why regions of our genome (assuming of course, that you are human!) that need to be transcribed very often such as promoter regions of highly expressed genes, are AT-rich, like the TATA box.
Problem 2: Formation of Secondary Structures
This point is very much tied to the first point. When GC-rich regions form secondary structures, particularly hairpin loops, they’re very stable, and so they stick around and accumulate. Furthermore, these secondary structures don’t melt well at usual PCR denaturation temperatures.
Additionally, primers used to amplify GC-rich regions tend to form self- and cross-dimers as well as stem-loop (or hairpin) structures that can impede the progress of the DNA polymerase along the template molecule leading to truncated PCR products. GC-rich sequences at the 3’ end of primers can also lead to mispriming.
What a disaster!
Poof! *the sounds of your confidence in your ability to do PCR deflating*
But wait! We have several solutions for you if you have problems amplifying GC-rich regions in your PCR!
5 Easy Solutions If You Are Having Problems Amplifying GC-rich Regions
Solution 1: Increasing Your Melting Temperature
This is a very sensible and easy solution (in theory, at least.) The higher your melting temperature, the more likely those troublesome secondary structures formed by GC-rich regions are to separate.
However, be wary as this can result in lower product yields! This is because your enzyme of choice, which is doing the amplification, can begin to denature more quickly at temperatures in excess of 92.5°C.
Therefore, it is advisable to only use higher melting temperatures for the first few cycles and to definitely avoid going over 95°C. It might take some playing around, but this solution is often a nice, easy starting point if you have problems amplifying GC-rich regions.
Solution 2: Adjusting Your Magnesium Concentration
Non-specific amplification, in general, can be exacerbated and even caused by using excessive concentrations of magnesium in your PCR reaction. Therefore, it’s a good idea to test the optimum concentration using gradient or titration PCR.
In a nutshell, the well or tube on the absolute left should contain less than what you think will work, and the well or tube on the absolute right should contain an excessive amount of magnesium. This allows a gradient to form in the middle so you can determine the lowest possible amount you can use to achieve your product.
Solution 3: Using Additives in Your PCR Reactions
Another way researchers can potentially improve problems in amplifying GC-rich regions is through the use of additives in their PCR reactions. These can include additives such as DMSO, glycerol, and BSA.
However, there are no hard and fast rules about these additives; their effects can be highly variable depending on the specific target, cycling condition, and the specific PCR enzyme and buffer being used for amplification.
Solution 4: Changing Your Polymerase or Buffer
So this solution is likely not to be the cheapest way to address your issues, but sometimes great science will require some fancy new products to be purchased!
Some companies have begun to develop buffers and polymerases specifically designed to amplify GC-rich regions. One such example is the OneTaq GC buffer from NEB. This buffer can be further supplemented with a high GC enhancer.
Other companies have optimized new polymerases to help amplify GC-rich regions. ThermoFisher has developed AccuPrime™ GC-Rich DNA Polymerase, which originates from the archaebacterium Pyrolobus fumarius.
This polymerase has increased processivity compared to Taq DNA polymerase. It remains active after 4 hours at 95°C, allowing you to combine this with our very first tip of increasing your melting temperature!
Solution 5: Changing Your PCR Method Entirely
Researchers have also developed entirely new ways to perform PCR on GC-rich templates in order to increase its success. One such example is known as Slow-down PCR. Slow-down PCR requires the addition of 7-deaza-2′-deoxyguanosine, a dGTP analog to the PCR mixture.
Slow-down PCR also uses a standardized cycling protocol with varying temperatures. Generally, ramp rates are lowered, and the PCR is run for additional cycles compared to a more standard PCR program. 
Problems Amplifying GC-rich Regions Summarized
So now you understand why amplifying GC-rich regions in your PCR reaction is causing you headaches, but most importantly, you’ve got plenty of options to address your problems!
Let us know what has worked for you in the comments below.
Originally published on May 26, 2015. Reviewed and updated June 2022.
- Kool ET. (2001) Hydrogen bonding, base stacking, and steric effects in DNA replication. Annu Rev Biophys Biomol Struct. 30:1–22.
- Frey U., Bachmann H., Peters J. et al. (2008) PCR-amplification of GC-rich regions: ‘slowdown PCR’. Nat Protoc 3, 1312–1317.