wild - type phi29 dnap ( 833,000 u ml ; specific activity 83,000 u mg ) was supplied by enzymatics corporation .
phosphoramidites ( including abasic residue ( dspacer ) , 3-spacer c3 cpg , and acridine ) were from glen research .
dna oligonucleotides were synthesized at the stanford university protein and nucleic acid ( pan ) facility .
lyophilized oligonucleotides received from pan were re - suspended in 7 m urea , 0.1 tbe , purified by denaturing polyacrylamide gel electrophoresis , and quantified using a nanodrop 1000 ( thermo scientific ) .
2 m each of a 70mer dna template ( supplementary fig . 1 ) and a 23-nt dna primer bearing a 5 fluorescein ( 6-fam ) label , were combined in the presence or absence of blocking oligomer ( fig 1b(ii ) ) at 2.4 m final concentration .
this mixture was incubated at 90 c for three minutes in 1x te , 100 mm kcl buffer , followed by slow - cooling to room temperature
. 1 m of preannealed dna substrate was then incubated for 5 hours at room temperature ( approximately 23c ) in nanopore buffer ( 22.5 l final volume ) supplemented with 10 mm mgcl2 , 0.75 m phi29 dnap , and 100 m dntps as indicated ( fig .
reactions were terminated by the addition of buffer - saturated phenol . following phenol / chloroform extraction and ethanol precipitation , dried dna pellets were dissolved in 7 m urea , 0.1x tbe and resolved by denaturing polyacrylamide gel electrophoresis .
gels were 17% acrylamide : bisacrylamide ( 19:1 ) , 7 m urea , 1 tbe , and run at 18 w. 6-fam labeled dna primer products were visualized on a uvp gel documentation device using a sybr gold filter .
setup of the nanopore device and insertion of an -hl nanopore into a lipid bilayer have been described .
briefly , a single -hl nanopore was inserted into a lipid bilayer that separates two wells that each contained 100 l of nanopore buffer ( ph 8) .
a 180 mv potential was applied across the bilayer and ionic current was measure through the nanopore between agcl electrodes in series with an integrating patch clamp amplifier ( axopatch 200b , molecular devices ) in voltage clamp mode .
data were recorded using an analog - to - digital converter ( digidata 1440a , molecular devices ) at 100 khz bandwidth in whole - cell configuration then filtered at 5 khz using a analogue low - pass bessel filter .
experiments were conducted at 23c with 1 m dna substrate preannealed to blocking oligomer in nanopore buffer with 0.75 m phi29 dnap , 10 mm mg , and 100 m of each dntp added to the nanopore cis well unless otherwise noted .
a single nanopore experiment is defined as the time during which ionic current data were acquired from one hl nanopore in an intact bilayer before termination by bilayer rupture , loss of channel conductance , or completion of a preset number of translocation events .
successful forward and reverse dna template translocations ranged for 50 to 500 per experiment .the ratio of fast ( < 200 ms ) dna alone events to polymerase - bound dna events was approximately 10 to 1 unless otherwise noted .
ionic current output from the nanopore device was analyzed using clampfit 10.2 software ( axon instruments ) after additional smoothing of data at 2 khz using a low - pass gaussian filter unless otherwise noted .
the standardized ionic current map in figure 3b was compiled from ten translocation events from the experiment in figure 2 .
these ten events each started at 23 pa , traversed two peaks at 35 pa , and ended by stalling at 23 - 24 pa .
ionic current steps within these ten translocation events were counted as discrete states if their durations were 3 ms or greater . for the data summarized in supplementary figure 4 , sequence - dependent pauses were scored if the duration of a discrete ionic current state between 0 and 16 was 200 ms or greater , or if fluctuations between any two discrete states between 0 and 16 was 200 ms or greater .
wild - type phi29 dnap ( 833,000 u ml ; specific activity 83,000 u mg ) was supplied by enzymatics corporation .
phosphoramidites ( including abasic residue ( dspacer ) , 3-spacer c3 cpg , and acridine ) were from glen research .
dna oligonucleotides were synthesized at the stanford university protein and nucleic acid ( pan ) facility .
lyophilized oligonucleotides received from pan were re - suspended in 7 m urea , 0.1 tbe , purified by denaturing polyacrylamide gel electrophoresis , and quantified using a nanodrop 1000 ( thermo scientific ) .
2 m each of a 70mer dna template ( supplementary fig . 1 ) and a 23-nt dna primer bearing a 5 fluorescein ( 6-fam ) label , were combined in the presence or absence of blocking oligomer ( fig 1b(ii ) ) at 2.4 m final concentration .
this mixture was incubated at 90 c for three minutes in 1x te , 100 mm kcl buffer , followed by slow - cooling to room temperature
. 1 m of preannealed dna substrate was then incubated for 5 hours at room temperature ( approximately 23c ) in nanopore buffer ( 22.5 l final volume ) supplemented with 10 mm mgcl2 , 0.75 m phi29 dnap , and 100 m dntps as indicated ( fig .
following phenol / chloroform extraction and ethanol precipitation , dried dna pellets were dissolved in 7 m urea , 0.1x tbe and resolved by denaturing polyacrylamide gel electrophoresis . gels were 17% acrylamide : bisacrylamide ( 19:1 ) , 7 m urea , 1 tbe , and run at 18 w. 6-fam labeled dna primer products were visualized on a uvp gel documentation device using a sybr gold filter .
setup of the nanopore device and insertion of an -hl nanopore into a lipid bilayer have been described . briefly , a single -hl nanopore was inserted into a lipid bilayer that separates two wells that each contained 100 l of nanopore buffer ( ph 8) .
a 180 mv potential was applied across the bilayer and ionic current was measure through the nanopore between agcl electrodes in series with an integrating patch clamp amplifier ( axopatch 200b , molecular devices ) in voltage clamp mode .
data were recorded using an analog - to - digital converter ( digidata 1440a , molecular devices ) at 100 khz bandwidth in whole - cell configuration then filtered at 5 khz using a analogue low - pass bessel filter .
experiments were conducted at 23c with 1 m dna substrate preannealed to blocking oligomer in nanopore buffer with 0.75 m phi29 dnap , 10 mm mg , and 100 m of each dntp added to the nanopore cis well unless otherwise noted .
a single nanopore experiment is defined as the time during which ionic current data were acquired from one hl nanopore in an intact bilayer before termination by bilayer rupture , loss of channel conductance , or completion of a preset number of translocation events .
successful forward and reverse dna template translocations ranged for 50 to 500 per experiment .the ratio of fast ( < 200 ms ) dna alone events to polymerase - bound dna events was approximately 10 to 1 unless otherwise noted .
ionic current output from the nanopore device was analyzed using clampfit 10.2 software ( axon instruments ) after additional smoothing of data at 2 khz using a low - pass gaussian filter unless otherwise noted .
the standardized ionic current map in figure 3b was compiled from ten translocation events from the experiment in figure 2 .
these ten events each started at 23 pa , traversed two peaks at 35 pa , and ended by stalling at 23 - 24 pa .
ionic current steps within these ten translocation events were counted as discrete states if their durations were 3 ms or greater . for the data summarized in supplementary figure 4 ,
sequence - dependent pauses were scored if the duration of a discrete ionic current state between 0 and 16 was 200 ms or greater , or if fluctuations between any two discrete states between 0 and 16 was 200 ms or greater .
 single - molecule techniques have been developed for commercial dna sequencing1,2 . 
 one emerging strategy uses a nanopore to analyze dna molecules as they are driven electrophoretically in single file order past a sensor3 - 5 . however , uncontrolled dna strand electrophoresis through nanopores is too fast for accurate base reads6 
 . a proposed solution would employ processive enzymes to deliver dna through the pore at a slower average rate7 . here , we describe forward and reverse ratcheting of dna templates through the hemolysin ( -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 . 
ONLINE METHODS
Materials
Bulk phase assay of DNA elongation and digestion by phi29 DNAP in the presence or absence of blocking oligomer
Nanopore experiments
Nanopore data analysis
Supplementary Material