It’s hard to imagine a molecular biology process that isn’t made more complicated by GC-rich DNA. From getting synthetic DNA constructs to sequencing, extreme GC content — that is, anything more than about 65% GC — can be a real hassle. The same is true for PCR amplification: extensive optimization is often needed to amplify GC-rich DNA templates. But these GC-rich regions play a crucial role in gene regulation, gene expression, genome functionality, and disease development, making them critical targets for biological experimentation.
To help address this challenge, we validated a technique using DMSO and betaine with our repliQa HiFi ToughMix® product that’s fast, inexpensive, and efficient. Even without the traditional enhancers, repliQa HiFi ToughMix is able to amplify GC content varying from 32% to 70% of a sequence. But with the addition of betaine or DMSO to improve the yield and specificity of high-GC templates without inhibiting polymerase activity, the approach can handle GC content as high as 82% with ease. This application note details the process and results in a sample experiment.
To validate the method, we used high molecular weight human genomic DNA as the template. Primers were designed to amplify specific regions in the human genome known to have high GC-content and associated with genetic diseases. These region-specific targets ranged from 149 base pairs to 281 base pairs, with GC content from 77% to more than 82%. These targets could not be amplified with standard PCR amplification techniques. Two of the targets, with GC content of about 78%, were successfully amplified with repliQa HiFi ToughMix alone.
Next, we tested whether the enhancers betaine and DMSO had any effect on the PCR amplification with repliQa HiFi ToughMix for all five targets. We found that both 1M betaine and 5% DMSO greatly improved amplification for our targets, but that the addition of betaine and DMSO together in the PCR mix was not effective. Consequently, we stopped testing the mixture of both DMSO and betaine. In the target with the highest GC content, only 5% DMSO was effective for amplification.
We conclude that this method will require testing with both DMSO and betaine to select the best enhancer for any specific GC-rich target. This fast and efficient technique could be extremely useful when studying high GC-regions that are difficult to amplify in biological and clinical studies.