Automated Forward and Reverse Ratcheting of DNA in a Nanopore at Five Angstrom Precision()

Single-molecule techniques have been developed for commercial DNA sequencing(1,2). One emerging strategy uses a nanopore to analyze DNA molecules as they are driven electrophoretically in single file order past a sensor(3-5). However, uncontrolled DNA strand electrophoresis through nanopores is too fast for accurate base reads(6). A proposed solution would employ processive enzymes to deliver DNA through the pore at a slower average rate(7). Here, we describe forward and reverse ratcheting of DNA templates through the α–hemolysin (α-HL) nanopore controlled by wild-type phi29 DNA polymerase (phi29 DNAP). DNA strands were examined in single file order at one nucleotide spatial precision in real time. The registry error probability (either an insertion or deletion during one pass along a template strand) ranged from 10% to 24.5% absent optimization. This general strategy facilitates multiple reads of individual template strands and is transferrable to other nanopore devices for implementation of DNA sequence analysis.