Anal. Chem. 2005, 77, 7090-7093
Design for DNA Separation Medium Using Bacterial Cellulose Fibrils Mari Tabuchi*,† and Yoshinobu Baba†,‡
Department of Molecular and Pharmaceutical Biotechnology, Graduate School of Pharmaceutical Science, The University of Tokushima COE, 1-78 Shomachi, Tokushima 770-8505, Japan, and Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, Nagoya 464-8603, Japan and National Institute of Advanced Industrial Science and Technology, Takamatsu 761-0395, Japan
In this paper, we present a novel DNA separation medium using bacterial cellulose fibrils. Bacterial cellulose has an intrinsic three-dimensional micrometer- to nanometerscale network structure. Addition of this material to a lowconcentration polymer solution (10 000 DPw), stiffness (Young module of a dry sheet is 33.3 GPa),14 and hydrogen-bond network.13,15,16 Many unique characteristics and applications of BC have been exploited. For example, it is a useful food additive (it is well known as a component of the “Nata de Coco”), and it is also utilized in construction of a commercially available vibration membrane in a speaker phone.17 In the current studies, we investigated the use of underivatized BC fragments as a component of an electrophoretic separation medium. Our design for this new medium is presented in Figure 1Bb. Two different types of mesh structure coexist in the medium: the mesh derived from the conventional polymer solution and that due to the BC fibrils. Thus, the structure is composed of ∼10-µm fragments containing 10-nm to 1-µm mesh with BC rigid fibrils and the several 10-nm mesh of the flexible polymer network. We speculated that this new structure would allow high-resolution separation of a wide range of DNA fragments and that it would be more effective than conventional highviscosity polymer solutions for the resolution of small DNAs including single-nucleotide polymorphisms (SNPs). Herein, we demonstrate the use of the novel nanostructure system using BC nanofibrils for the separation of biomolecules. (11) Huang, M.-F.; Kuo, Y.-C.; Huaang, C.-C.; Chang, H.-T. Anal. Chem. 2004, 76, 192-196. (12) Brown, A. J. J. Chem. Soc. 1886, 49, 432-439. (13) Cousins, S. K.; Brown, R. M., Jr. Polymer 1997, 38, 903-912. (14) Tabuchi, M.; Watanabe, K.; Morinaga, Y,; Yoshinaga, F. Biosci, Biotechnol. Biochem. 1998, 62, 1451-1454. (15) Kuga, S.; Takagi, S.; Brown, R. M., Jr. Polymer 1993, 34, 3293-3297. (16) Javis, M. Nature 2003, 426, 611-612. (17) Yamanaka, S.; Watanabe, K.; Kitamura, N.; Iguchi, M.; Mitsuhashi, S.; Nishi, Y.; Uryu, M. J. Mater. Sci., 1989, 25, 3141-3145. 10.1021/ac0511389 CCC: $30.25
© 2005 American Chemical Society Published on Web 09/30/2005
Figure 1. (A) Diagrams of sieving matrixes. (a) Small mesh size in a conventional high-concentration polymer solution; (b) wide mesh size in a diluted polymer solution (right, ultradiluted polymer solution); (c) nanospace in the nanostructures; (d) mixture of diluted polymer solution and nanospheres; (e) double-mesh concept. (B) (a) SEM images of BC medium; (b) schematic diagram for separation in BC medium.
EXPERIMENTAL PROCEDURE Bacterial Cellulose. A BC buffer was prepared in the following manner. BC was produced by a static culture of Acetobacter hansenii (ATCC 700178 Sumisho Pharmal International). The wet pellicle contained ∼0.1% (dry weight per total wet pellicle weight) cellulose fibrils. Approximately 10-µm fragments of BC pellicle were prepared using a sterile mill (SM-1 Labcat) from wet pellicles under wet conditions. BC fibrils consist of nanometer-scale-thickness network frame and 10-nm to 1-µm pore size. This BC suspension is used as an additive to the medium (BC medium). Contamination of bacteria DNA from BC medium was not detected. A polymer solution (hydroxypropylmethyl cellulose; HPMC, Sigma) was utilized for the control medium. To investigate the effect of additional media, a BC medium (1-3 wt % BC fragments) was added to the control medium. DNA Samples. In Figure 2, a 1 µg/µL solution of 10-bp dsDNA ladder (Invitrogen, 10 -bp intervals fragments, 100 and 330 bp are higher), a 1 µg/µL solution of Φx174-HincII digest (Takara Biomedicals, Siga, Japan), and a 100-bp DNA ladder (Takara) were utilized. For long DNAs, 1 ng/µL solution of a 2-Log DNA ladder (Bio Labs, New England; 100 bp -8 kbp; 500 bp is composed of 500 and 517 bp, and the peaks of 500, 1000, and 3000 bp are higher), and a 100-15-kbp DNA ladder (Takara) are analyzed. In Figure 4 in the text, the resolution of each peak of 10/20 bp in Figure 2a, 500/517 bp in Figure 2d, and 12/15 bp in Figure 2e were calculated. Each DNA sample (1 µL) was dissolved in 9 µL of Milli-Q water (ICN Biomedicals, Aurora, OH). A polymer solution in an i-chip kit (0.7% HPMC, Hitachi Chemical, pH 9), was used as the running buffer for microchip electrophoresis. Lower is for BC and upper is for control for each figure.
SNP Analysis. We also proved the BC medium for another biochemical application, namely, detection of SNPs in the electrophoresis. Single-strand conformation polymorphism (SSCP) in DNA (Ki-ras genes) was analyzed. The SNPs samples were prepared by PCR (Gene AMP PCR System 9700; Applied Biosystems) using templates for the Ras mutant c-Ki-ras codon 12 (Takara Bio, 108 bp), ras gene primer prepared by FAM (Klabo, Osaka, Japan), Tag, dNTP mixture, and PCR buffer in a PCR amplification kit (Takara Bio). The PCR protocol involved 20 cycles, half that used in the conventional protocol, with each cycle consisting of 40 °C for 1 min, 55 °C for 2 min, and 72 °C for 1 min. The ssDNA was analyzed by the SSCP method. PCR samples were diluted 10-fold with distilled water, denatured at 94 °C for 3 min, and then immediately put into an ice/water bath and kept for 5 min. Detection System. A microchip electrophoresis analysis system (SV1100, Hitachi Electronics, Hitachi Japan), with a blue LED (470 nm) light source was used as the basic detection method. DNAs were separated in the microchannel using 750 V of separation voltage (220 V/cm). A microchip with a microchannel 100 µm wide and 30 µm deep (i-chip3 or i-chip12, Hitachi Chemical, Hitachi, Japan) was used. RESULTS AND DISCUSSION Proposed Addition of BC Fibrils to Electrophoresis Buffer. The BC medium was composed of 0.3% (w/v) BC and 0.49% (w/v) HPMC. The control medium contained 0.49% HPMC alone. Figure 2 shows electropherograms of DNA separated in the new medium and in conventional medium. The 10-bp ladder, which has peaks at intervals of 10 bp between 10 and 330 bp, was completely resolved in the BC medium (lower panel in Figure Analytical Chemistry, Vol. 77, No. 21, November 1, 2005
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Figure 3. Electropherograms of SNPs analysis in BC medium. The wild type (a, GGT) and mutants (b, GCT; c, CGT; d, TGT; e, AGT; f, GTT) prepared by PCR from ras mutant c-Ki-ras codon 12 (TakaraBio, Sigma) and mixtures of wild type and each mutant (g-j) are shown. 1-4: peak identification number.
Figure 2. Electropherograms of DNA in BC medium and in conventional medium. (a) 10-bp dsDNA ladder (10-bp interval fragments, 100 and 330 bp are higher), (b) Φx174 HincII digest, (c) 100bp DNA ladder (Takara), (d) 2-Log DNA ladder (Bio Labs; 100 bp-8 kbp, 500 bp is composed of 500 and 517 bp, and the peaks of 500, 1000, and 3000 bp are higher), and (e) 100-15-kbp DNA ladder (Takara). Upper trace shows the electropherogram by conventional medium and lower shows that by the BC medium for each panel.
2a). In particular, there is excellent separation below 100 bp, even though the concentration of polymer (0.49% HPMC) is lower than usual. The same concentration of HPMC without BC resulted in lower resolution (upper panel in Figure 2a) because the separation of small DNA normally requires a high concentration of polymer 7092 Analytical Chemistry, Vol. 77, No. 21, November 1, 2005
solution (e.g., g1% HPMC). The 291- and 297-bp peaks in the Φx174-HincII-digested standards were also separated with higher resolution by the BC medium than the control (lower panel in Figure 2b; Rs ) 1.5 and 0.5, respectively). In addition, the 900and 1000-bp peaks were well-resolved in the 100-bp DNA ladder using the BC medium (lower panel in Figure 2c, Rs ) 1.5). The BC medium allowed the separation of DNAs as long as 15 kbp (Figure 2d and e). Application to the SSCP Analysis. We further investigated the application of BC fragments in SSCP analysis. Figure 3 shows the electropherograms of Ki-ras gene separation. Remarkably, this medium allowed determination of SSCP for SNP analysis, even though it has a viscosity of
