Abstract
Nanopore technologies are being developed for fast and direct sequencing of single DNA molecules through detection of ionic current modulations as DNA passes through a pore's constriction1,2. Here we demonstrate the ability to resolve changes in current that correspond to a known DNA sequence by combining the high sensitivity of a mutated form of the protein pore Mycobacterium smegmatis porin A (MspA)3 with phi29 DNA polymerase (DNAP)4, which controls the rate of DNA translocation through the pore. As phi29 DNAP synthesizes DNA and functions like a motor to pull a single-stranded template through MspA, we observe well-resolved and reproducible ionic current levels with median durations of ∼28 ms and ionic current differences of up to 40 pA. Using six different DNA sequences with readable regions 42–53 nucleotides long, we record current traces that map to the known DNA sequences. With single-nucleotide resolution and DNA translocation control, this system integrates solutions to two long-standing hurdles to nanopore sequencing2.
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Acknowledgements
We thank M. Akeson and G.M. Cherf for getting us started with the blocking oligomer phi29 DNAP technique, sharing their CAT DNA and reading the manuscript. We thank J. Bartlett and G. Nayler for their help running experiments and D. Feldman for writing data acquisition code. This work was supported by the US National Institutes of Health, National Human Genome Research Institute $1000 Genome Program Grants R21HG004145, R01HG005115 and R01HG006321.
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E.A.M., I.M.D. and J.H.G. conceptualized the project. E.A.M., I.M.D., A.H.L. and J.H.G. designed the experiments, wrote the paper and contributed equally. E.A.M., I.M.D., A.H.L., K.W.L., M.K.H., N.G. and J.H.G. analyzed the data. K.W.L., M.K.H. and N.G. collected data. M.P. and M.N. produced the MspA mutants. J.H.G. supervised the project.
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Manrao, E., Derrington, I., Laszlo, A. et al. Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase. Nat Biotechnol 30, 349–353 (2012). https://doi.org/10.1038/nbt.2171
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DOI: https://doi.org/10.1038/nbt.2171
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