DNA sequencing by capillary electrophoresis using copolymers of acrylamide andN,N-dimethyl-acrylamide

Abstract

Copolymers of acrylamide (AM) and N,N-dimethylacrylamide (DMA) with AM to DMA molar ratios of 3:1, 2:1 and 1:1 and molecular weights of about 2.2 MDa were synthesized. The polymers were tested as separation media in DNA sequencing analysis by capillary electrophoresis (CE). The dynamic coating ability of polydimethylacrylamide (PDMA) and the hydrophilicity of polyacrylamide (PAM) have been successfully combined in these random copolymers. A separation efficiency of over 10 million theoretical plates per meter has been reached by using the bare capillaries without the additional polymer coating step. Under optimized separation conditions for longer read length DNA sequencing, the separation ability of the copolymers decreased with decreasing AM to DMA molar ratio from 3:1, 2:1 and 1:1. In comparison with PAM, the copolymer with a 3:1 AM:DMA ratio showed a higher separation efficiency. By using a 2.5% w/v copolymer with 3:1 AM:DMA ratio, one base resolution of 0.55 up to 699 bases and 0.30 up to 963 bases have been achieved in about 80 min at ambient temperatures.

Critical factors for high-performance physically adsorbed (dynamic) polymeric wall coatings for capillary electrophoresis of DNA

“…Table 2 lists the compositions of the DEA/DMA copolymers used in this study. 1 H NMR analysis shows that DEA content in the DEA/DMA copolymers is close to the original comonomer composition, which indicates that the reactivity of the comonomers were very similar under our reaction conditions [27]. Hence, random copolymers were formed.…”

“…Polymers that have been investigated as adsorptive polymeric wall coatings for microchannel electrophoresis include poly(N,N-dimethylacrylamide) (PDMA) [23], polyvinylpyrrolidone (PVP) [24], and a variety of other novel, acrylamide-based polymers [2,[25][26][27]. At this time, PDMA and PVP are the most widely used adsorptive wall coatings for biomolecule analysis.…”

Influence of fluorophor dye labels on the migration behavior of polymerase chain reaction - amplified short tandem repeats during denaturing capillary electrophoresis

“…The higher hydrophilicity of polyacrylamide vs. pDMA may be responsible for the fact that the fluorophor dye-dependent anomalies are less pronounced in classical sequencing gels. Perhaps, the influence of dye-specific effects in CE can be effectively suppressed by using polymers, which are more hydrophilic (e.g., linear polyacrylamide or copolymers of acrylamide and N,N-dimethylacrylamide [25,26]) than the entangled pDMA polymer which was used in our study as the sieving matrix in CE. …”

Separation of double-stranded DNA fragments by capillary electrophoresis using polyvinylpyrrolidone and poly(N,N-dimethylacrylamide) transient interpenetrating network

“…Some copolymers have been developed and tested as DNA separation media. They can be divided into three categories according to the type:(i) random copolymers, e.g., copolymer of poly-N,N-diethylacrylamide (PDEA) and PDMA [23,24], polyacrylamide (PAM)-co-PDMA [25]; (ii) block copolymers, e.g., PEG end capped with fluorocarbon [19]; (iii) graft copolymers, e.g., polyacrylamide grafted with poly-N-isopropylacrylamide (PAM-g-PNi-PAM) [26], PEO grafted with N-isopropylacrylamide (PEOg-PNiPAM). Recently, noncross-linked interpenetrating polymer networks (IPNs) of PAA and PVP, which combined the sieving ability of PAA and the coating ability of PVP [27], was used for dsDNA analysis in our laboratory.…”