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

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 region

SUBMITTER: Manrao EA 

PROVIDER: S-EPMC3757088 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature