Sparsely cross‐linked “nanogels” for microchannel DNA sequencing

We have developed sparsely cross‐linked “nanogels”, sub‐colloidal polymer structures composed of covalently linked, linear polyacrylamide chains, as novel DNA sequencing matrices for capillary electrophoresis. The presence of covalent cross‐links affords nanogel matrices with enhanced network stability relative to standard, linear polyacrylamide (LPA), improving the separation of large DNA fragments. Nanogels were synthesized via inverse emulsion (water‐in‐oil) copolymerization of acrylamide and N,N ‐methylenebisacrylamide (Bis). In order to retain the fluidity necessary in a replaceable polymer matrix for capillary array electrophoresis (CAE), a low percentage of the Bis cross‐linker (< 10 −4 mol%) was used. Nanogels were characterized by multiangle laser light scattering and rheometry, and were tested for DNA sequencing by CAE with four‐color laser‐induced fluorescence (LIF) detection. The properties and performance of nanogel matrices were compared to those of a commercially available LPA network, which was matched for both weight‐average molar mass ( M w ) and extent of interchain entanglements ( c/c *). Nanogels presented in this work have an average radius of gyration of 226 nm and a weight‐average molar mass of 8.8×10 6 g/mol. At concentrations above the overlap threshold, nanogels form a clear, viscous solution, similar to the LPA matrix ( M w ∼ 8.9×10 6 g/mol). The two matrices have similar flow and viscosity characteristics. However, because of the physical network stability provided by the internally cross‐linked structure of the nanogels, a substantially longer read length (∼ 63 bases, a 10.4% improvement) is obtained with the nanogel matrix at 98.5% accuracy of base‐calling. The nanogel network provides higher‐selectivity separation of ssDNA sequencing fragments longer than 375 bases. Moreover, nanogel matrices require 30% less polymer per unit volume than LPA. This is the first report of a sequencing matrix that provides better performance than LPA, in a side‐by‐side comparison of polymer matrices matched for M w and extent of interchain entanglements.