DNA sequencing by microchip electrophoresis using mixtures of high- and low-molar mass poly(N,N-dimethylacrylamide) matrices

• Published in: Electrophoresis Vol. 29; no. 23; pp. 4663 - 4668
• Main Authors: Hert, Daniel G, Fredlake, Christopher P, Barron, Annelise E
• Format: Journal Article
• Language: English
• Published: Weinheim Wiley-VCH Verlag 01.12.2008; WILEY-VCH Verlag; WILEY‐VCH Verlag
• Subjects: Acrylamides - chemistry; DNA - chemistry; DNA - genetics; DNA sequencing; Electrophoresis, Microchip - methods; Humans; Microchip electrophoresis; Microfluidics; Mixed molar mass; Molecular Weight; N-dimethylacrylamide; Poly(N; Poly(N,N‐dimethylacrylamide); Sequence Analysis, DNA - methods; Viscosity
• ISSN: 0173-0835; 1522-2683
• DOI: 10.1002/elps.200800389

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.