The EU-funded GENOTYPING NANOPORES (Genotyping using solid-state nanopores and pepetide nucleic acid markers – a new tool for single-molecule molecular diagnostics) project has developed a revolutionary, low-cost, single-molecule genotyping method based on nanopore sensing of peptide nucleic acids (PNAs). Nanopore analysis involves using a voltage to drive molecules through a nanoscale pore in a membrane between two electrolytes. Changes in the current can be measured as it passes single molecules in the nanopore. Scientists involved in GENOTYPING NANOPORES previously showed that PNAs were detectable using tiny solid-state nanopores. They have now improved nanopore fabrication, the signal over noise of the measurements, and the biomolecular strategies for efficient PNA invasion of the nanopore. For high specificity sensing, the scientists used γPNA probes for high affinity with DNA. Results show that the ion-current signal corresponding to the passage of a double stranded DNA molecule on which three γPNA molecules were bound displays three distinct features. This enables straightforward identification and quantification of the DNA molecules as they translocate through the pore. Researchers calibrated the distance between any two PNA molecules in base pairs. In real-time, they then identified and classified genes in the two HIV subtypes having over 92 % similarity. The nanopore classification method gave a rapid and highly accurate discrimination and quantification of the two HIV variants. Extending the approach in the final phase of the project, the researchers created a more general method for DNA barcoding. Using light-emitting 'molecular beacons' to colour code sequence-specific probes, the fluorophore of the next beacon emits photons on unzipping, which are collected by a sensitive microscope. This procedure is compatible with single-stranded DNA (ssDNA) and ssRNA molecules and has been published in the prestigious journal Nano Letters, 15, 745-752, 2015. The impact on society promises to be tremendous and means opening up multiple possibilities for real-life applications in the biomedical field. Besides early detection of cancer in circulating tumour DNA, these methods can be used to rapidly identify antimicrobial-resistant pathogens and optimise antibiotic administration. Further development of the technology will mean a low-cost, portable high-throughput device for a broad range of genomic diagnostics.
Nanopore, DNA sequencing, peptide nucleic acids, DNA barcoding, molecular beacons