Hairpin structure facilitates multiplex high-fidelity DNA amplification in real-time PCR

Abstract

Effective polymerase chain reactions (PCR) are important in bio-laboratories. It is essential to detect rare DNA-sequence variants for early cancer diagnosis or for drug-resistance mutations identification. Some of the common detection quantitative PCR (qPCR) methods are restricted in the limit of detection (LoD) because of the high polymerase misincorporation rate in Taq DNA polymerases. High-fidelity (HiFi) DNA polymerases have a 50- to 250-fold higher fidelity. Yet, there are currently no proper designs for multiplexed HiFi qPCR reactions. Moreover, the popularity of targeting highly multiplex DNA sequences requires minimizing PCR side products, as the potential of dimerization grows quadratically as the plexes of primers increases. Efforts tried before were either an add-on step, or technology-specific, or requiring high-level computing skills. There lacks an easy-to-apply and cost-effective method for dimerization reduction. Here, we presented the Occlusion System, composed of a 5’-overhanged primer and a probe with a short-stem hairpin. We demonstrated that it allowed multiplexing high-fidelity qPCR reaction, it was also compatible with the current variant-enrichment method to improve the LoD by 10-fold. Further, we found that the Occlusion System reduced the dimerization up to 10-fold in highly multiplexed PCR. Thus, the Occlusion System satisfactorily improved both qPCR sensitivity and PCR efficiency.

Cost-Efficient Sequence-Based Nonextensible Oligonucleotide in Real-Time PCR and High-Throughput Sequencing

“…Various mixtures of genomic DNA NA18562 and NA18537 with different variant allele fractions (VAFs) were input in the BDA assay targeting one single nucleotide polymorphism (SNP) loci using Taq or high-fidelity DNA polymerase (Figure S12). The high-fidelity DNA polymerase with NEO BDA shows an LoD about 0.01% VAF, the Taq DNA polymerase with NEO BDA shows an LoD around 0.1%, consistent with previous claims. , It revealed that NEO blockers would not affect the LoD of BDA methods.…”

“…On the basis of the steric effect hypothesis, we first appended a 4nt stem and 4nt loop hairpin sequence to the 3′ region of another sequence that could perfectly bind to the targeted genomic region. The size of the hairpin stem was designed to be short and thus less supportive for polymerase binding to reduce unnecessary polymerase occupancy . The hairpin-attached oligo was then input into the single-plex qPCR reaction as the forward primer.…”

“…The high-fidelity DNA polymerase with NEO BDA shows an LoD about 0.01% VAF, the Taq DNA polymerase with NEO BDA shows an LoD around 0.1%, consistent with previous claims. 12,28 It revealed that NEO blockers would not affect the LoD of BDA methods. An mBDA 80-plex 27 panel targeting sequence-selective PCR amplification of SNP alleles was used with the NEO blocker (Figure 4 and Table S6).…”

“…The size of the hairpin stem was designed to be short and thus less supportive for polymerase binding to reduce unnecessary polymerase occupancy. 28 The hairpin-attached oligo was then input into the single-plex qPCR reaction as the forward primer. block its extension, the modified forward primer would not produce amplification.…”

“…Though the phosphorothioate bond (PTO) has been reported to resist the proofreading feature of high-fidelity DNA polymerase before, its blocking efficiency is not comparable with dual internal C3 spacers. Notably, high-fidelity DNA polymerases reveal significant advantages in reducing polymerase error, , and they have been reported to improve detection sensitivity when integrated with previous methods. ,− Thus, it is urged to have more affordable blocking options available for high-fidelity DNA polymerases.…”