Separation of 4‐color DNA sequencing extension products in noncovalently coated capillaries using low viscosity polymer solutions

Abstract

A low viscosity (ca. 75cP) solution using polydimethylacrylamide (PDMA) was developed for separating DNA sequencing extension products by capillary electrophoresis (CE). This medium gave a length-of-read (LOR) value of approximately 600 bases in about 2 h using four-color sequencing in 50 microm capillary at 42 degrees C under a field of 160 V/cm. This medium also works in bare capillaries by noncovalently coating the surface to suppress both electroosmotic flow (EOF) and DNA-capillary wall interactions, and eliminates the need for complicated covalent coatings. At least 100 successive sequencing runs were performed in the same capillary by simply pumping fresh medium after every run, without requiring any reconditioning of the capillary surface between runs. The thermal stability of the noncovalent coating can be improved by adding small amounts of high molecular weight PDMA to the separation medium. The advantages of low viscosity separation media and uncoated capillaries are of paramount importance to develop high-throughput instruments for DNA sequencing.

Experimental study of the effect of disorder on DNA dynamics in post arrays during electrophoresis

“…Neutral, adsorbed polymers suppress electroosmotic flow (EOF) during electrophoresis by forming a dynamic surface coating [23]. While several polymers have been shown to reduce EOF in capillaries [24], we chose PVP based on its success in microfluidic devices [1,5,6,14,18,20,22,25,26]. The device can be conditioned with the polymer overnight to develop a uniform surface coating [22,27,28]; however, several groups have had success conditioning the device for less than one minute [24], or simply including polymer in the running buffer [6,14,20,25,26].…”

Characterization of low viscosity polymer solutions for microchip electrophoresis of non-denatured proteins on plastic chips

“…Hydrophobic polymers such as methylcellulose (MC) which do not adsorb strongly onto normal capillary surfaces are very well adapted to coat hydrophobic surfaces of polymeric microchips. [10][11][12] More hydrophobic polymers such as poly(vinylpyrrolidone) (PVP) or poly(N,N-dimethylacrylamide) (PDMA) form more stable adsorbed coatings, but are more likely to interact with hydrophobic patches on the surface of folded proteins which reduces separation efficiency. 13 For this report, we chose microchips made of poly(methyl methacrylate) (PMMA) which is one of the most common polymeric substrates for fabrication of microchips and it has been applied for protein separation.…”

Theoretical Study of Comb Polymers Adsorption on Solid Surfaces

“…These oligomers need to be present in the solution at a high concentration, in order to yield sufficient dynamic coating. Presently, the most efficient applications of dynamic coating have involved Poly-Dimethyl Acrylamide (PDMA) [12] or copolymers of this polymer with other acrylic monomers [13]. This polymer seems to present an interesting affinity to silica walls, thanks to the presence of hydrogen bonding, while remaining soluble enough in water to behave as a sieving matrix.…”