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

The report discusses the impact of GC-rich regions on the amplification efficiency of neighboring amplicons in various PCR techniques, including nanoplate digital PCR (ndPCR), real-time quantitative PCR (qPCR), and droplet digital PCR (ddPCR). The study found that amplification efficiency decreased progressively with proximity to a GC-rich region, with the nearest amplicon yielding only 9% of the reference value in ndPCR.

The effect was mitigated by physical separation of amplicons using restriction enzyme digestion or additives such as DMSO and betaine. In qPCR, the effect on quantitation depended on whether the standard curve material and the sample shared similar neighboring GC-rich sequence context. The findings suggest that GC-rich regions can have broad practical implications for PCR-based quantitation, especially in regions like mammalian promoters, enhancers, and other gene regulatory elements.

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