Endonuclease IV-mediated substrate structure allosteric for universal and sensitive mutant allele enrichment and discrimination

In this work, the structure allosteric induced by Endonuclease IV (Endo IV) preference to the specific substrate was ingeniously combined with the asymmetric PCR technique. As a result, it could construct a universal and straightforward strategy for mutant allele enrichment (IVME) to improve the sensitivity of downstream mutation detection methods. At the initial time, both the mutant-type target (MT) and the wild-type target (WT) would hybridize with the probe (FP) containing apurinic/apyrimidinic site to form a flap structure, which could not be used to perform asymmetric PCR. Once Endo IV was added, the substrate formed by FP and MT, not the FP-WT, could be recognized and cleaved due to the high single nucleotide discrimination ability of Endo IV. Subsequently, the flap structure at the 3′ terminal was destroyed and exposed an active 3′-OH, which could act as a primer in the asymmetric PCR. Thus, both the absolute amount and the abundance of MT were enhanced. 1% and 0.01% MT could be directly distinguished by coupling with Sanger sequencing and Endo IV-assisted nucleic probe hybridization-based DNA mutation detection method, respectively. EGFR T790M mutation in the peripheral blood of a non-small cell lung cancer patient was detected by IVME coupled with Sanger sequence. The results were also verified by next-generation sequencing, which demonstrated the potential clinical application of our proposed strategy.