DNA sequencing by microchip electrophoresis using mixtures of high‐ and low‐molar mass poly(<i>N,N</i>‐dimethylacrylamide) matrices

TLDR

Mixed molar‑mass polymer matrices improve DNA fragment separation compared to single‑molar‑mass matrices. This study systematically investigates how varying proportions of high‑ and low‑molar‑mass pDMA polymers affect sequencing performance in microfluidic chips. Two polydisperse pDMA samples (3.5 MDa and 770 kDa) were blended at different fractions while keeping the total polymer concentration at 5 % w/v. High‑molar‑mass polymer is required for read lengths >400 bases, yet a 3 % 3.5 MDa + 2 % 770 kDa blend yields similar >520‑base reads while reducing viscosity fivefold, showing that long reads can be achieved in a low‑viscosity tuned blend.

Abstract

Previous studies have reported that mixed molar mass polymer matrices show enhanced DNA sequencing fragment separation compared with matrices formulated from a single average molar mass. Here, we describe a systematic study to investigate the effects of varying the amounts of two different average molar mass polymers on the DNA sequencing ability of poly(N,N-dimethylacrylamide) (pDMA) sequencing matrices in microfluidic chips. Two polydisperse samples of pDMA, with weight-average molar masses of 3.5 MDa and 770 kDa, were mixed at various fractional concentrations while maintaining the overall polymer concentration at 5% w/v. We show that although the separation of short DNA fragments depends strongly on the overall solution concentration of the polymer, inclusion of the high-molar mass polymer is essential to achieve read lengths of interest (>400 bases) for many sequencing applications. Our results also show that one of the blended matrices, comprised of 3% 3.5 MDa pDMA and 2% 770 kDa pDMA, yields similar sequencing read lengths (>520 bases on average) to the high-molar mass matrix alone, while also providing a fivefold reduction in zero-shear viscosity. These results indicate that the long read lengths achieved in a viscous, high-molar mass polymer matrix are also possible to achieve in a tuned, blended matrix of high- and low-molar mass polymers with a much lower overall solution viscosity.