Dehai Liang

Copolymer solutions as separation media for DNA capillary electrophoresis

A review on copolymers used as DNA separation media in capillary electrophoresis is presented. Copolymers can combine the desirable properties of different monomers, yielding many attractive features, such as high sieving ability, low viscosity, self-assembly behavior and dynamic coating ability. Copolymers with different molecular architecture, including block copolymers, random copolymers, and graft copolymers, have been developed and tested as DNA separation media with unique and tailored properties that cannot be achieved easily by using only homopolymers.

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

A noncross‐linked interpenetrating polymer network (IPN), consisting of poly(N,N‐dimethylacrylamide) (PDMA) and polyvinylpyrrolidone (PVP, weight‐average molecular weight Mw = 1×106 g/mol) was synthesized by polymerizing N,N‐dimethylacrylamide (DMA) monomers directly in PVP buffer solution and tested as a separation medium for double‐stranded (ds)DNA analysis without further purification. Due to the incompatibility of PVP and PDMA, a simple solution mixture could incur a microphase separation and showed poor performance on dsDNA separation. However, a dramatic improvement was achieved by the formation of an IPN. We attributed the high sieving ability of IPN as due to an increase in the number of entanglements by the more extended polymer chains. Apparent viscosity studies showed that the IPN had a much higher viscosity than the simple mixture containing the same amount of PDMA and PVP. In 1×Tris‐borate‐EDTA (TBE) buffer, the concentration ratio of PDMA and PVP had a great effect on the DNA separation. At optimal conditions, the 22 fragments in pBR322/HaeIII DNA were successfully separated within 15 min, with a resolution of better than 1.0 for 123/124 bp.

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

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 2.2u2005MDa 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 80u2005min at ambient temperatures.

Separation of double-stranded DNA fragments by capillary electrophoresis in interpenetrating networks of polyacrylamide and polyvinylpyrrolidone

Mixtures of two polymers with totally different chemical structures, polyacrylamide and polyvinylpyrrolidone (PVP) have been successfully used for double‐stranded DNA separation. By polymerization of acrylamide in a matrix of PVP solution, the incompatibility of these two polymers was suppressed. Laser light scattering (LLS) studies showed that highly entangled interpenetrating networks were formed in the solution. Further systematic investigation showed that double‐stranded DNA separation was very good in these interpenetrating networks. With a concentration combination of as low as 2% w/v PVP (weight‐average molecular mass Mr = 1×106 g/mol) + 1% w/v polyacrylamide (Mr = 4×105 g/mol), the 22 fragments in pBR322/HaeIII DNA, including the doublet of 123/124 bp, have been successfully separated within 6.5 min. Under the same separation conditions, similar resolution could only be achieved by using polyacrylamide (Mr = 4×105 g/mol) with concentrations higher than 6% w/v and could not be achieved by using only PVP (Mr = 1×106 g/mol) with a concentration as high as 15% w/v. It is noted that the interpenetrating network formed by 2% PVP and 1% polyacrylamide has a very low viscosity and can dynamically coat the inner wall of a fused‐silica capillary. The separation reached an efficiency of more than 107 theoretical plate numbers/m and a reproducibility of less than 1% relative standard deviation of migration time in a total of seven runs. The interpenetrating network could stabilize polymer chain entanglements. Consequently, the separation speed was increased while retaining resolution.

DNA sequencing by capillary electrophoresis using mixtures of polyacrylamide and poly(N, N-dimethylacrylamide)

The possibility of using polymer mixtures with different chemical compositions as a DNA sequencing matrix by capillary electrophoresis (CE) has been exploited. Polyacrylamide (PAM, 2.5%, w/v) having a molecular mass of 2.2 x 10(6) has been mixed with poly(N,N-dimethylacrylamide) (PDMA) having molecular masses of 8000, 470000 and 2.1 x 10(6) at concentrations of 0.2, 0.5 and 1% (w/v). Unlike polymer mixtures of the same polymer with different molecular masses, the use of polymer mixtures with different chemical compositions encounters an incompatibility problem. It was found that the incompatibility increased with increasing PDMA molecular mass and PDMA concentration, which resulted in decreased efficiency in DNA sequencing. Also, the incompatibility had a more pronounced effect on the efficiency as the base number was increased. However, by choosing a low-molecular-mass PDMA of 8000 and a low concentration of 0.2% (w/v), the incompatibility of PAM and PDMA has been alleviated. At the same time, the advantage of using polymer mixtures revealed a higher efficiency for such a polymer mixture when compared with PAM. The mixture also endowed the separation medium with a dynamic coating ability. An efficiency of over 10 x 10(6) theoretical plates per meter has been achieved by using the bare capillaries without the additional chemical coating step.

Effect of glycerol-induced DNA conformational change on the separation of DNA fragments by capillary electrophoresis

The effect on DNA separation of adding glycerol to the running buffer was studied using linear polyacrylamide (LPA) or poly(ethylene oxide) (PEO) as separation medium. For both LPA and PEO, it was found that the addition of 25% (v/v) glycerol to the running buffer enhanced the separation of large double-stranded DNA fragments and increased the migration time. The two buffers used, 1 x TBE (Tris-boric acid-EDTA) and 1 x TTE (Tris-N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS)-EDTA), showed similar improvement, but the effect on the 1 x TBE buffer was more amplified. The difference in buffer properties, such as viscosity, conductance, and pH, had little effect on the separation. We attribute the improvements made in the separation to the ability of glycerol to induce a conformational change in DNA as demonstrated by dynamic light scattering results. The presence of glycerol can increase the electrostatic interactions between the phosphate groups, decrease the hydration sphere of the polynucleotides, and compete with water to form hydrogen bonds with the side group of bases. These interactions increase the DNA contour length and reduce the effective charge over weight ratio, which can explain the experimental data. The complex formed by boric acid and glycerol had a stronger effect on the DNA conformation change than glycerol itself. This enhancement was also observed in DNA sequencing analysis.

Clay-enhanced DNA separation in low-molecular-weight poly(N,N-dimethylacrylamide) solution by capillary electrophoresis.

The effect of the separation medium in capillary electrophoresis consisting of a low‐molecular‐mass poly(N,N‐dimethylacrylamide) (PDMA) solution on the DNA separation by adding a small amount of montmorillonite clay into the polymer matrix is presented. On the separation of the pBR322/HaeIII digest, both the resolution and the efficiency were increased by adding 2.5–5.0×10–5g/mL clay into the 5% w/v PDMA with a molecular mass of only 100 K. Moreover, there was no increase in the migration time of DNA fragments. Similar results were observed by using a C‐terminated pGEM‐3Zf(+) sequencing DNA sample in a sequencing buffer. Experimental data also showed that the addition of clay increased the viscosity of the polymer solution. We attribute this effect to the structural change of the polymer matrix caused by the exfoliated clay sheets, whereby the thin clay sheets function like a “dynamic cross‐linking plate” for the PDMA chains and effectively increase the apparent molecular mass of PDMA.

Spatial open-network formed by mixed triblock copolymers as a new medium for double-stranded DNA separation by capillary electrophoresis

A mixture of two polyoxybutylene‐polyoxyethylene‐polyoxybutylene (BEB) triblock copolymers (B6E46B6 and B10E271B10, respectively) was used as a new separation medium for separating double‐stranded DNA (dsDNA) fragments by capillary electrophoresis (CE). The two block copolymer mixtures were designed to form mixed flower‐like micelles in dilute solution and a homogeneous gel‐like open‐network with hydrophobic clusters as cross‐linking points at higher polymer concentrations. Being a polyoxyalkylene block copolymer gel, the separation medium has some special advantages, including the temperature‐dependent sol‐gel transition that makes sample injection easy, and the self‐coating of the inner capillary wall that makes experimental procedures simple and reproducible. Furthermore, it can shorten the elution time and further improve the separation resolution, especially for small dsDNA fragments, when compared with EPE‐type separation media, e.g., F127 (E99P69E99, with P being polyoxypropylene) block copolymer gels formed by the closed packing of spherical micelles. Single base pair resolution can be achieved by using the new separation medium for dsDNA fragments up to over 100 base pairs.

Mixed triblock copolymers used as DNA separation medium in capillary electrophoresis

A polymer solution, formed by mixing two polyoxybutylene-polyoxyethylene-polyoxybutylene (BEB) triblock copolymers (B10E270B10 and B6E46B6), was tested as a new separation medium for double-stranded DNA separation in capillary electrophoresis. The mixture of B10E270B10 and B6E46B6 has a viscosity-adjustable property and a dynamic coating ability, which makes the medium very easy to handle. The performance of the mixture on the DNA separation is greatly affected by the mass ratio of the two constituents. There is a minimum amount of concentration for B10E270B10, below which the medium will lose its performance. The addition of B6E46B6 increases both the selectivity and the separation efficiency. The optimal concentration, with 3% (w/v) B10E270B10 and 5% (w/v) B6E46B6, is determined with the consideration of both speed and resolution. A resolution of 1.3 was achieved on the separation of 123/124 base pairs in the pBR322/HaeIII digest within 20 min by using a 10 cm column of 75 microm I.D., demonstrating the potential use of mixtures of amphiphilic block copolymers as an effective DNA separation medium.

Formation of concentration gradient and its application to DNA capillary electrophoresis

A new method to introduce the concentration gradient into the capillary has been developed and its application to DNA capillary electrophoresis is presented. The concentration gradient produced by mixing 5% w/v polyacrylamide‐co‐poly(N‐dimethylacrylamide) (PAM‐co‐PDMA) solution and 1 × Tris/N‐tris(hydroxymethyl)methyl‐3‐amino‐propanesulfonic acid/EDTA (TT) + 5u2005M urea buffer was successfully achieved by using two programmable syringe pumps with strict control of dead volume, flow rate, and pressure balance. This method has the advantages of high stability, reproducibility, and versatility. The column with concentration gradient greatly improved the resolution, especially for the large DNA fragments, due to a decrease in band width broadening with time. A column containing 2—4% w/v gradient in four steps had a longer read length, shorter separation time and better resolution (after 380 base) than that of 4% w/v single concentration polymer solution. The number of steps in the gradient had almost no effect on the performance. The change in the average concentration by relocating the position of the same step gradient, i.e., a combination of different low concentration to high concentration polymer solution ratios, resulted in a different migration time, read length and resolution.