E. A. Manrao, I. M. Derrington, M. Pavlenok, M. Niederweis, and J. H. Gundlach.
Nucleotide discrimination with DNA immobilized in the MspA
nanopore.
PLoS One, 6(10):e25723, 2011.
MANR2011-AA
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 -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.