A 265-base DNA sequencing read by capillary electrophoresis with no separation matrix

Abstract

Electrophoretic DNA sequencing without a polymer matrix is currently possible only with the use of some kind of "drag-tag" as a mobility modifier. In free-solution conjugate electrophoresis (FSCE), a drag-tag attached to each DNA fragment breaks linear charge-to-friction scaling, enabling size-based separation in aqueous buffer alone. Here we report a 265-base read for free-solution DNA sequencing by capillary electrophoresis using a random-coil protein drag-tag of unprecedented length and purity. We identified certain methods of protein expression and purification that allow the production of highly monodisperse drag-tags as long as 516 amino acids, which are almost charge neutral (+1 to +6) and yet highly water-soluble. Using a four-color LIF detector, 265 bases could be read in 30 min with a 267-amino acid drag-tag, on par with the average read of current next-gen sequencing systems. New types of multichannel systems that allow much higher throughput electrophoretic sequencing should be much more accessible in the absence of a requirement for viscous separation matrix.

Figure 1

Electrophoretic analysis of the purity of increasing lengths of protein drag-tags; the cluster of peaks at 7 minutes is “free” unconjugated DNA, and the second peak is the DNA-drag-tag conjugate. Measured alpha values are noted. (A) 27mer (204-aa) and 36mer (267-aa) proteins conjugated to M13 sequencing primer, post-single-base extension reaction (18-nt oligomer). (B) 54mer (390-aa) and 72mer (516-aa) proteins conjugated to 30-nt oligomer. Electrophoresis is performed on ABI 3100 (36-cm capillary) with electrokinetic injection at 22 V/cm for 20 seconds, separation at 312 V/cm, 55 °C, in 1 X TTE buffer with 7 M urea and 1:200 dilution of POP-6™ as a wall coating.

Figure 2

Four-color sequencing electropherogram with 36mer drag-tag (267-aa); 265 bases are resolved by electrophoresis without a sieving polymer under same conditions as in Figure 1. M13mp18 template is “read” backwards, starting at the right of the bottom panel.

Figure 3

Peak width (FWHM) is plotted versus length of DNA sequencing fragment. At any length of DNA, peak width increases as drag-tag size increases.

Figure 4

Separation peak analyses of a series of sequencing separations performed at 9 electric field strengths (E = 62-312 V/cm, applied voltage of 3-15 kV). Plate height H is plotted versus reciprocal speed for the (A) 27mer and (B) 36mer protein drag-tag. H was measured for two lengths of DNA, 61 bases (C-terminated) and 104 bases (A-terminated). Dashed line is the linear fit of H vs u^-1 data for all but the highest E.

Figure 5

Separation factor S versus DNA length from sequencing data with the 36mer drag-tag are compared at varied E (155-312 V/cm, fragments are considered well-resolved if S ≤ 1). The highest field strength gave the lowest S and is predicted to give the longest sequencing read.