PMC

Current trace for polymerase synthesis. (a) Illustration of phi29 DNAP during synthesis. (b) Typical current trace for synthesis of ‘block homopolymer’ DNA. At the beginning of the read, multiple thymines (dT₄) produce a current level of I_b/I_o ~ 0.20. At the end of the read, two abasic residues (XX) produced a high current (I_b/I_o ~ 0.61). *, toggles. (c) Mean currents of levels extracted from b are plotted with the associated DNA sequence.

Reading a repetitive DNA template. (a) Example trace for a DNA template composed of repeated ‘CAT’ trinucleotides, with the exception of one ‘CAG’ triplet in the middle of the sequence. (b) Mean currents of levels extracted from a with the associated DNA sequence are shown. The current trace exhibits a repeating pattern of three levels (blue bars) interrupted by the single dG substitution (highlighted in orange). Four levels are affected by the single dG with the largest deviations closest to the substitution. This indicates that the residual current is principally influenced by one or two nucleotides with a lesser influence from neighboring nucleotides.

Reading heteromic DNA. (a) Example event for heteromer DNA 1. Unzipping and synthesis phases are indicated. (b) Demonstration of the repeatability and similarity of current levels found in multiple events. Time-ordered levels for n = 20 events collected on N = 2 pores are found using a level detection algorithm. A consensus current level sequence was created from multiple events. The time-ordered levels consistent with the consensus sequence are overlaid (one color for each event). The symmetry of the plot about level ± 1 shows that the same levels occur during both unzipping and synthesis (in reverse order). The correlated sequence is shown below. Levels not detected, owing to repeat nucleotides and/or similar currents, are indicated with the unobserved nucleotide beneath the sequence. (c) s.d. derived from the scatter of each overlaid level. The average 1-sigma fluctuation in levels is below 1% of I_o. (d) The probability, P_obs, of finding a given level in the proper order. Some levels, such as level 10 or level 21 are found in all events (n = 20). Levels immediately before the transition from unzipping to synthesis are often too fast to be detected, resulting in low P_obs.

Event structure. (a) Crystal structure of M2-NNN MspA. Charged vestibule residues are indicated in blue (negative) or red (positive). (b) A schematic depicting a standard experiment. Roman numerals correspond to positions in the current trace in c. (c) The measured blockage current (I_b) as a fraction of the open pore current (I_o) is shown for a sample event. (i) A single MspA pore (purple) in a lipid bilayer (gray). The template strand (black) contains the sequence to be read. A primer strand (blue) is hybridized to the template’s 3′ end. A blocking oligomer (red) with a 3′ end of several abasic sites is adjacent to the primer. The phi29 DNAP (green) binds to the DNA to form a complex that is driven into MspA. A positive voltage is applied to the trans side. The single stranded 5′ end of the DNA-motor complex threads through MspA and the ionic current drops. (ii) The electric force on the captured strand draws the DNA through the phi29 DNAP, unzipping the blocking oligomer. Arrows show the direction of motion of the DNA template strand. The ionic current exhibits distinct steps while nucleotides pass through the pore. (iii) The blocking oligomer is removed and DNA reverses direction (marked by blue dashed line). (iv) The phi29 DNAP incorporates nucleotides into the primer strand, pulling the template toward the cis side. The current repeats previously observed levels in reverse time-order. Two abasic sites produce a high current peak (~0.6 I_o) indicated by red Xs. This marker is first seen during unzipping and then again during synthesis. When synthesis is complete, the DNA and DNAP escape to the cis volume, marked by the return to I_o.