Nanopore Filter: A Method for Counting and Extracting Single DNA Molecules Using a Biological Nanopore

Abstract

This paper describes a method for the real-time counting and extraction of DNA molecules at the single-molecule level by nanopore technology. As a powerful tool for electrochemical single-molecule detection, nanopore technology eliminates the need for labeling or partitioning sample solutions at the femtoliter level. Here, we attempt to develop a DNA filtering system utilizing an α-hemolysin (αHL) nanopore. This system comprises two droplets, one filling with and one emptying DNA molecules, separated by a planar lipid bilayer containing αHL nanopores. The translocation of DNA through the nanopores is observed by measuring the channel current, and the number of translocated molecules can also be verified by quantitative polymerase chain reaction (qPCR). However, we found that the issue of contamination seems to be an almost insolvable problem in single-molecule counting. To tackle this problem, we tried to optimize the experimental environment, reduce the volume of solution containing the target molecule, and use the PCR clamp method. Although further efforts are still needed to achieve a singlemolecule filter with electrical counting, our proposed method shows a linear relationship between the electrical counting and qPCR estimation of the number of DNA molecules.

Lipid vesicle-based molecular robots

“…Since the detection sensitivity of nanopore sensing largely depends on the size and structure of the pores, a wide range of proteins have been investigated for the detection of specific targets. 60,64 In 1996, α-hemolysin (αHL), with a pore size of 1.4 nm at the restriction, which is compatible with the translocation of single-stranded DNA (ssDNA), was initially employed by Kasianowicz et al for detecting polynucleotides. 65 In 2010, Mycobacterium smegmatis porin A (MspA) was found to be suitable for discriminating individual nucleotide bases.…”