Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase

Nat Biotechnol. 2012 Mar 25;30(4):349-53. doi: 10.1038/nbt.2171.

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 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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • DNA-Directed DNA Polymerase / chemistry
  • DNA-Directed DNA Polymerase / genetics
  • High-Throughput Nucleotide Sequencing / instrumentation*
  • High-Throughput Nucleotide Sequencing / methods*
  • Nanopores*
  • Nucleotides / chemistry
  • Nucleotides / genetics
  • Porins / chemistry
  • Porins / genetics

Substances

  • Nucleotides
  • Porins
  • mspA protein, Mycobacterium smegmatis
  • DNA-Directed DNA Polymerase