DNA sequencing by microchip electrophoresis using mixtures of high- and low-molar mass poly(N,N-dimethylacrylamide) matrices.
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.
Hert
Daniel G
DG
Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA.
Fredlake
Christopher P
CP
Barron
Annelise E
AE
D000178:Acrylamides / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000235:genetics; D046209:Electrophoresis, Microchip / Q000379:methods*; D006801:Humans; D044085:Microfluidics; D008970:Molecular Weight; D017422:Sequence Analysis, DNA / Q000379:methods*; D014783:Viscosity
D000178:Acrylamides; C429790:poly(N,N-dimethylacrylamide); D004247:DNA