Amplifying GC rich regions can be troublesome and its useful to understand why its so and what can help.

Amplifying GC rich content sometimes require optimizing PCR, adding additives, or designing new primers. Many manufacturers produce PCR kits for high GC content. But, sometimes these commercial kits are unable to do their duty.

 

GC rich region is difficult to work with due to their strong affinity. Nucleic acids that build gene are composed of A T G and C bases. DNA is double stranded in structure as A complements T and G pairs with C. A and T bind with two hydrogen bonds, but G and C are connected with 3 bonds. This makes GC bond stronger making it need more energy to separate the strand. Higher temperature during PCR can release hydrogen bond. However, when G and C are in long consecutive sequence or many GC sequence close together, high temperature alone may not suffice. Also, long GC sequence might lead to secondary structure. The key to successfully amplifying high GC content is to get annealing temperature where no secondary structure is formed but nucleic acid can still complement.

PCR optimization such as using step down PCR might help in such amplification. This method use higher annealing temperature at the first few cycles then lowering it for the rest of the cycles. Though it is meant for getting specific PCR product, it is also applicable for temperate GC content.

Additives such as DMSO, betaine, formamide, or 7-deaza-GTP when added to PCR reaction reduce the annealing temperature required. DMSO is the commonly used additive and is recommended at a concentration of 1% to start with. You may go up to 2.5% to see if that helps. All these reagents can reduce the formation of secondary structure. From my experience, combination of reagents can be more effective. However, it is necessary to try various reagent concentrations and combination in PCR to get the optimum results.

A new primer design can also be decisive in getting PCR product. Pick new site of primer to either shorten or extend the product so the GC content will similar to AT content.