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. 2010 Sep 30;4(3):32210.
doi: 10.1063/1.3479997.

DNA separation by cholesterol-bearing pullulan nanogels

Keisuke Kondo, Noritada Kaji, Sayaka Toita, Yukihiro Okamoto, Manabu Tokeshi, Kazunari Akiyoshi, Yoshinobu Baba

DNA separation by cholesterol-bearing pullulan nanogels

Keisuke Kondo et al. Biomicrofluidics. .

Abstract

We present an application of a novel DNA separation matrix, cholesterol-bearing pullulan (CHP) nanogels, for microchip electrophoresis. The solution of the CHP showed a unique phase transition around 30 mg∕ml and formed gel phase over this critical concentration. This gel phase consists of the weak hydrophobic interactions between the cholesterols could be easily deformed by external forces, and thus, loading process of the CHP nanogels into microchannels became easier. The high concentration of the CHP nanogels provided excellent resolutions especially for small DNA fragments from 100 to 1500 bp. The separation mechanism was discussed based on Ogston and Reptation models which had developed in gels or polymer solutions. The result of a single molecule imaging gave us an insight of the separation mechanism and the nanogel structures as well.

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Figures

Figure 1
Figure 1
Chemical structures of cholesterol modified pullulans and schematic illustration of the formation of a nanogel by self-association of the cholesteryl groups in water.
Figure 2
Figure 2
Photographs of the CHP nanogel solution at various concentration in the vials.
Figure 3
Figure 3
Separation of 100 bp DNA ladder in various concentration of the CHP solution. All the electric field strength for separation was 92 V∕cm and the effective separation length was 30 mm.
Figure 4
Figure 4
Dependence of the electrophoretic mobility of 100 bp DNA ladder on (a) the CHP concentration and (b) the reciprocal of DNA size. Each data point represents the average of three to nine electropherograms.
Figure 5
Figure 5
Migration behavior of a single DNA molecule in the CHP solution at 30 mg∕ml. The applied electric field was 18 V∕cm. The kink part of the DNA molecule indicated by the white arrows migrated for the first 3 s, and then, the U-shape was resolved by pulling the short arm.
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