GC-rich regions affect amplification efficiency of neighboring amplicons in qPCR and digital PCR.

DNA polymerase chain reaction (PCR) has been widely used for the quantitation of DNA and RNA samples. Multiple factors can affect amplification efficiency, including GC-rich regions. The proximity effects of GC-rich regions on the amplification efficiency of neighboring amplicons that are not themselves GC-rich are not fully understood. Using a GFP-6.0Kb recombinant adeno-associated virus (rAAV) vector plasmid containing a 300 bp region with 79% GC content, we evaluated the effect of this GC-rich region on six amplicons (38-61% GC) at varying distances from it in nanoplate digital PCR (ndPCR), real-time quantitative PCR (qPCR), and droplet digital PCR (ddPCR). In ndPCR, amplification efficiency decreased progressively with proximity to the GC-rich region, with the nearest amplicon yielding only 9% of the reference value. Restriction enzyme digestion that physically separated amplicons from the GC-rich region restored efficiency to approximately 100%. In qPCR, the effect on quantitation depended on whether the standard curve material and the sample shared similar neighboring GC-rich sequence context. The effect was not observed in ddPCR under the conditions tested. Additives including DMSO and betaine, and physical separation by restriction enzyme digestion or sonication, mitigated the effect in ndPCR and qPCR. These findings may have broad practical implications for PCR-based quantitation, as GC-rich regions are prevalent in many mammalian promoters, enhancers, and other gene regulatory elements.