Nanolock–Nanopore Facilitated Digital Diagnostics of Cancer Driver Mutation in Tumor Tissue

Abstract

Cancer driver mutations are clinically significant biomarkers. In precision medicine, accurate detection of these oncogenic changes in patients would enable early diagnostics of cancer, individually tailored targeted therapy, and precise monitoring of treatment response. Here we investigated a novel nanolock−nanopore method for single-molecule detection of a serine/threonine protein kinase gene BRAF V600E mutation in tumor tissues of thyroid cancer patients. The method lies in a noncovalent, mutation sequence-specific nanolock. We found that the nanolock formed on the mutant allele/probe duplex can separate the duplex dehybridization procedure into two sequential steps in the nanopore. Remarkably, this stepwise unzipping kinetics can produce a unique nanopore electric marker, with which a single DNA molecule of the cancer mutant allele can be unmistakably identified in various backgrounds of the normal wild-type allele. The single-molecule sensitivity for mutant allele enables both binary diagnostics and quantitative analysis of mutation occurrence. In the current configuration, the method can detect the BRAF V600E mutant DNA lower than 1% in the tumor tissues. The nanolock−nanopore method can be adapted to detect a broad spectrum of both transversion and transition DNA mutations, with applications from diagnostics to targeted therapy.

Design and MinION testing of a nanopore targeted gene sequencing panel for chronic lymphocytic leukemia

“…Today, other single-molecule platforms are also being developed in order to overcome the limit of the nanopore error rate and detect very low-frequencies point mutations 40 , 41 . In the future, the implementation of these innovative approaches in MinION sequencing may contribute to better discriminate between mutated and wild-type DNA molecules and will be extended to achieve the concurrent detection of multiple biomarker mutations.…”

Next-Generation Sequencing in Acute Lymphoblastic Leukemia

“…Indeed, the main MinION challenge to be dealt with is the high error rate especially in homopolymeric sequences, for which basecalling is a crucial step; thus, several basecallers are being developed, trying to improve read accuracy [142], as well as new bioinformatic pipelines for variant calling analysis [137,143]. Indeed, studies of other single-molecule sequencing technologies are ongoing, attempting to defeat the MinION error rate [144,145]. In the future the integration of these advanced technologies in nanopore sequencing will likely yield a true technologic improvement that will bring advanced genomic analysis within everyone’s reach.…”

Recognition of Single‐Point Mutation Using a Biological Nanopore

“…On the other hand, nanopore sensing is able to directly detect the translocation of short DNA strands via the blocking current of the open‐pore state. [ 18 ] Regarding the point mutation analysis of short DNA using nanopore sensing, several approaches have been reported, including using an enzyme reaction, [ 19 ] nanolock, [ 20 ] DNA nanopore, [ 21 ] or probe DNA. [ 22,23 ] Although these methods can determine the presence or absence of a single mutation, prediction of the mutation position has remained challenging.…”