Nucleotide Discrimination with DNA Immobilized in the MspA Nanopore

Nanopore sequencing has the potential to become a fast and low-cost DNA sequencing platform. An ionic current passing through a small pore would directly map the sequence of single stranded DNA (ssDNA) driven through the constriction. The pore protein, MspA, derived from Mycobacterium smegmatis, has a short and narrow channel constriction ideally suited for nanopore sequencing. To study MspA's ability to resolve nucleotides, we held ssDNA within the pore using a biotin-NeutrAvidin complex. We show that homopolymers of adenine, cytosine, thymine, and guanine in MspA exhibit much larger current differences than in α-hemolysin. Additionally, methylated cytosine is distinguishable from unmethylated cytosine. We establish that single nucleotide substitutions within homopolymer ssDNA can be detected when held in MspA's constriction. Using genomic single nucleotide polymorphisms, we demonstrate that single nucleotides within random DNA can be identified. Our results indicate that MspA has high signal-to-noise ratio and the single nucleotide sensitivity desired for nanopore sequencing devices

Measuring Single-Molecule DNA Hybridization by Active Control of DNA in a Nanopore

We present a novel application of active voltage control of DNA captured in a nanopore to regulate the amount of time the DNA is available to molecules in the bulk phase that bind to the DNA. In this work, the control method is used to measure hybridization between a single molecule of DNA captured in a nanopore and complementary oligonucleotides in the bulk phase. We examine the effect of oligonucleotide length on hybridization, and the effect of DNA length heterogeneity on the measurements. Using a mathematical model, we are able to deduce the binding rate of complementary oligonucleotides, even when DNA samples in experiments are affected by heterogeneity in length. We analyze the lifetime distribution of DNA duplexes that are formed in the bulk phase and then pulled against the pore by reversing the voltage. The lifetime distribution reveals several dissociation modes. It remains to be resolved whether these dissociation modes are due to DNA heterogeneity or correspond to different states of duplex DNA. The control method is unique in its ability to detect single-molecule complex assembly in the bulk phase, free from external force and with a broad (millisecond-to-second) temporal range