Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase
By E. Manrao, I. Derrington, A. Laszlo, Kyle W. Langford, Matthew K Hopper, N. Gillgren, Mikhail Pavlenok, M. Niederweis, J. Gundlach. Nature Biotechnology, 2012
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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 constriction. 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) with phi29 DNA polymerase (DNAP), 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 sequencing.
Subfields: Biomedical Engineering, Computational Mechanics, Electrical and Electronic Engineering
View Paper: https://www.semanticscholar.org/paper/bc205aa338bd362879f57691e85f488bf3f65a42
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Citations in the corpus
- Nanopore sequencing technology, bioinformatics and applications (2021, background): “Indeed, this motor protein provided the last piece of the puzzle; in February 2012, two groups demonstrated processive recordings of ionic currents for single-stranded DNA molecules that could be resolved into signals from individual nucleotides by combining phi29 DNA polymerase and a nanopore ( α -”
- The Third Revolution in Sequencing Technology. (2018, background): “However, due to technical obstacles the first successful sequencing results were reported only in 2012 [22].”
- Nanopore-based Fourth-generation DNA Sequencing Technology (2015, methodology): “demonstrated a new method to sequence ssDNA at single-nucleotide resolution using a mutant MspA nanopore and phi29 DNA polymerase [96], which can control the rate of DNA translocation with median durations of 28 ms and ionic current differences up to 40 pA.”
- From squiggle to basepair: computational approaches for improving nanopore sequencing read accuracy (2018, background): “Problematically, the translocation speed of motor proteins is generally nonuniform, disrupting the relationship between homopolymer length and detection time [24 – 26], a problem that has also been reported by ONT [23].”
- Three decades of nanopore sequencing (2016, background): “Given an effective sensing length of ~1.2 nm for the sensing region, it is not surprising that up to five neighboring bases simultaneously influence the current levels as a single DNA strand traverses through MspA’s limiting aperture 24,46 .”