The Cause of Your Problems

Problem - 1. Stability: Thermal and Structural

GC-rich DNA sequences are more stable than sequences with low GC-content. For PCR, this means that the higher the GC content, the higher the melting point of the DNA. Under pressure, such as when exposed to heat, the GC-rich sequences can take far more abuse than GC-low sequences. GC-rich DNA sequences are more stable than sequences with low GC-content, but, contrary to popular belief, the hydrogen bonds are not the primary reason for this stability. Stabilization is mainly due to stacking interactions called base stacking. There is some beautiful biochemistry and biophysics behind this and if you’re interested is a fantastic explanation as to why this stacking occurs.

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. These don’t melt well at usual PCR denaturation temperatures. Additionally, primers used to amplify GC-rich regions have a tendency to form self- and cross-dimers as well as stemloop 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.

SOLUTIONS

Solution 1. Increasing Your Melting Temperature

This is a very sensible solution (in theory.) The higher the temperature, the more likely those troublesome secondary structures formed by GC-rich regions are to separate. However this results in lower product yields as your taq begins to denature more quickly at temperatures in excess of 92.5°C, so it is advisable to only use higher melting temperatures for the first few cycles and to avoid going over 95°C. It might take some playing around but this is solution is a good starting point.

Solution 2. Adjusting Your Magnesium Concentration

Non-specific amplification in general can be exacerbated and even caused by using excessive concentrations of magnesium (Mg) so test the optimum concentration using gradient or titration PCR. In a nutshell, your well on the absolute left contain a less than what you think will work and your well on the absolute right contain an excessive amount of magnesium with a gradient in the middle so you can determine the lowest possible amount you can use to achieve your product.

Solution 3.

I found a few recipes online that various labs have found worked well for them:

Addition of 3-10% dimethyl sulfoxide (DMSO), most often 5%

Addition of 1M betaine and 5% DMSO – these may reduce your enzyme activity so add extra enzyme

Addition of betaine (Sigma), DMSO, DTT and BSA

Addition of ethylene glycol with 1,2-propanediol

Addition of 5 % DMSO and 1.25% formamide or just formamide

Addition of 5 – 10% absolute glycerol and higher temperature of annealing

Use regular HotStart and add 2M betaine

Use Kapa’s HiFi Hot Start enzyme

Use KAPA3G GC rich Kit with 5% DMSO

GC enhancers available in various kits like this Clontech, Epicentre and Qiagen also both offer kits for this problem if you’ve some money to spare.

Solution 4. Other types of PCR

Nested-PCR: I’ve written entirely on this technique so stay tuned for that!

Slowdown-PCR: explored here.

Hot-Start PCR.

Long range PCR: clontech and qiagen both offer kits for this problem if you’ve some money to spare.

Hope this info will help you.

Good luck!

Thank you very much @ Karen for your suggestions.

Phusion have GC specific buffers and enzyme., and it worked very well for my expts