Replication of Individual DNA Molecules under Electronic Control Using a Protein Nanopore

Nanopores can be used to analyse DNA by monitoring ion currents as individual strands are captured and driven through the pore in single file order by an applied voltage. Here we show that serial replication of individual DNA templates can be achieved by DNA polymerases held at the α-hemolysin nanopore orifice. Replication is blocked in the bulk phase, and is initiated only after the DNA is captured by the nanopore. We used this method, in concert with active voltage control, to observe DNA replication catalyzed by bacteriophage T7 DNA polymerase (T7DNAP) and by the Klenow fragment of DNA polymerase I (KF). T7DNAP advanced on a DNA template against an 80 mV load applied across the nanopore, and single nucleotide additions were measured on the millisecond time scale for hundreds of individual DNA molecules in series. Replication by KF was not observed when this enzyme was held atop the nanopore orifice at 80 mV applied potential. Sequential nucleotide additions by KF were observed upon controlled voltage reversals.