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Rapid sequencing gel electrophoresis using glycerol-tolerant sodium taurine medium
Analytical Biochemistry 390 (2009) 100–101
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Rapid sequencing gel electrophoresis using glycerol-tolerant sodium taurine medium Ken Hirano *, Tomomi Ishido, Mitsuru Ishikawa Health Technology Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu, Kagawa 761-0395, Japan
a r t i c l e
i n f o
Article history: Received 23 March 2009 Available online 11 April 2009
a b s t r a c t We describe a new glycerol-tolerant sodium taurine (ST) medium for rapid sequencing gel electrophoresis by substituting the standard conductive media of Tris–boric acid–ethylenediaminetetraacetic acid (EDTA) (TBE) and Tris–taurine–EDTA (TTE) and other low-ionic-strength media of sodium boric acid (SB). Low-ionic-strength and cost-effective ST media gave glycerol tolerance up to 50% (v/v) glycerol-containing DNA sample solution, shorter running time, and better resolution to separate small DNA oligonucleotides (20–45 mer) in 12% denaturing sequencing gel electrophoresis. Ó 2009 Elsevier Inc. All rights reserved.
Sequencing gel electrophoresis is an essential tool to analyze not only DNA sequences but also DNA polymerase for characterization of new recombinant enzymes [1], the incorporation activity of 20 -deoxyribonucleoside 50 -triphosphate (dNTP)1 derivatives for mutagenic effects and single-molecule sequencing [2,3], and inhibitor assays for molecular medicine [4]. The standard conducting media of Tris–boric acid–ethylenediaminetetraacetic acid (EDTA) (TBE) and Tris–taurine–EDTA (TTE) used for sequencing gel electrophoresis have a disadvantage in that a high voltage cannot be applied due to the high ionic strength of the media [5]. A long running time of gel electrophoresis is required, therefore, and is achieved by using standard media and a long-sized sequencing gel. A novel low-ionicstrength sodium boric acid (SB) medium was recently shown to shorten the running time and improve the resolution of DNA fragments in not only an agarose gel system [6] but also a sequencing gel system to analyze small DNA fragments (90–300 bp) [7]. In the current study, we found that SB medium was affected by glycerol in the reaction sample. Some DNA polymerases required a high concentration of glycerol in the reaction buffer (e.g., mammalian DNA polymerase-b required reaction buffer containing 15% [v/v] glycerol) [8]. Direct sample loading to the gel was needed for quantitative primer extension assay without DNA purification for DNA polymerase requiring a high concentration of glycerol and for commercial DNA polymerases stored in buffer containing 50% (v/v) glycerol. In the current study, we used new low-ionic-strength sodium taurine (ST) medium and evaluated glycerol-tolerant conducting media by
* Corresponding author. Fax: +81 87 869 3569. E-mail address: [email protected] (K. Hirano). 1 Abbreviations used: dNTP, 20 -deoxyribonucleoside 50 -triphosphate; EDTA, ethylenediaminetetraacetic acid; TBE, Tris–boric acid–EDTA; TTE, Tris–taurine–EDTA; SB, sodium boric acid; ST, sodium taurine; DIG, digoxygenin; PAGE, polyacrylamide gel electrophoresis; APS, ammonium persulfate. 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.04.006
substituting SB media for ST media for rapid sequencing gel electrophoresis in the presence of glycerol in the loading samples. The sequencing gel electrophoresis equipment used was the Sequi-Gen GT system (Bio-Rad, Hercules, CA, USA). As a glycerol-tolerant medium, ST medium was prepared at the final concentrations of 5 mM sodium hydroxide and 11 mM taurine (first grade, >98.5%, Sigma–Aldrich Japan, Tokyo, Japan) at pH 9.0. Comparison media of SB (10 mM sodium hydroxide [pH 8.5] adjusted with boric acid) [6], TBE (89 mM Tris, 89 mM boric acid, and 2 mM EDTA) [9], and TTE (89 mM Tris, 29 mM taurine [2-aminoethanesulfonic acid], and 0.1 mM EDTA) [9] were used. Digoxygenin (DIG)-labeled oligonucleotides (20–45 mer) were synthesized and purchased from Sigma–Aldrich Japan and analyzed on 12% denaturing polyacrylamide gel. Gels were 0.4 mm thick and 50 cm long. Different concentrations of glycerol (0–50%, v/v) in the DNA sample solution were tested in each conductive medium. Power during the electrophoresis was fixed at 50 W. Voltage, current, and temperature were recorded at the start and end of electrophoresis. After electrophoresis, separated DNA fragments were detected by chemiluminescence with a DIG detection system [2]. Chemiluminescent images were captured using X-ray film. Fig. 1 shows the separation of small oligonucleotides (20– 45 mer) using four conductive media (ST, SB, TTE, and TBE) in 12% denaturing sequencing gel electrophoresis at different glycerol concentrations (0–50%, v/v). A range of excess glycerol concentrations up to 50% (v/v) was selected to indicate the effect of glycerol. ST and TTE of taurine-based media gave glycerol-tolerant separation up to 50% (v/v) glycerol in high concentration. SB and TBE of boric acid-based media were affected by band blurring that was dependent on the glycerol concentration in the sample solution. In SB medium, bending in the sequencing tracks and shrinkage of the width of bands were observed, resulting in desirable resolution in the presence of glycerol in sequencing gel electrophoresis. The effect of glycerol in the boric acid-based
Notes & Tips / Anal. Biochem. 390 (2009) 100–101
101
Fig. 1. Analysis of small DNA fragments containing glycerol in sample solutions using various conductive media in 12% denaturing polyacrylamide sequencing gel electrophoresis. The DNA sample solution was a mixture of oligonucleotides of 20, 21, 22, 23, 24, 25, 30, 35, 40, and 45 mer. The concentrations of glycerol in DNA sample solutions were 0, 5, 10, 20, 30, 40, and 50% (v/v). Running time was established when the distance between 20 and 45 mer for the 0% glycerol sample was 11 cm for each conductive medium. Power during the electrophoresis was fixed at 50 W. Voltage, current, and temperature were recorded at the start and end of gel electrophoresis.
medium was due to the interaction of glycerol with boric acid in conductive media to form anionic esters, which migrate through the gel during electrophoresis [9]. When replacing boric acid in the gel and in the electrophoresis buffer with taurine, the latter suppressed interaction of glycerol in conductive media because of a weak aminosulfonic acid with a pKa of 9.06, similar to the pKa of boric acid (9.24) [9]. ST, as a taurine-based medium similar to TTE medium, suppressed the effect of glycerol interaction at the extreme end of glycerol concentration in the current study. ST medium gave not only glycerol tolerance but also rapid separation. The low ion conduction of ST medium also enabled the application of high voltage (by Ohm’s law) at a constant power of 50 W during gel electrophoresis. Application of high voltage reduced the running time of gel electrophoresis by 26% (119 min instead of 160 min) per run. The effect of this reduction may be important in sequencing gel electrophoresis because a long run time is necessary. The difference in temperature rise between each medium was small because the current used during electrophoresis between each medium was not greatly different (Fig. 1). The current in polyacrylamide gel electrophoresis (PAGE) is derived from not only the conducting media but also ammonium persulfate (APS) in the gel. This is different from agarose gel electrophoresis. APS, being a salt, may have affected the current to some extent and seems to affect the current difference in temperature rise. ST medium and SB medium are cost-effective; their reagent costs are 10-fold less expensive compared with Tris-based standard media [5]. In conclusion, we found that ST medium was glycerol tolerant and gave rapid separation of DNA fragments in sequencing gel electrophoresis. Glycerol-tolerant ST medium may also enable high-throughput analysis because of not only rapid separation but also labor-free sample loading on the sequencing gel without previous purification of reaction samples to remove glycerol. The
current study is important for developing a method for glyceroltolerant rapid analysis of DNA and RNA fragments using not only standard sequencing gel electrophoresis and native PAGE (which have been used for single-strand conformation polymorphisms) but also capillary and microfluidic chip-based electrophoresis for high-throughput analysis. Acknowledgment This work was supported by a grant from the Industrial Technology Research Program from the New Energy and Industrial Technology Development Organization (NEDO), Japan. References [1] N. Shimazaki, K. Yoshida, T. Kobayashi, S. Toji, K. Tamai, O. Koiwai, Overexpression of human DNA polymerase lambda in E. Coli and characterization of the recombinant enzyme, Gene Cells 7 (2002) 639–651. [2] A.S. Kamath-Loeb, A. Hizi, H. Kasai, L.A. Loeb, Incorporation of the guanosine triphosphate analog 8-oxo-dGTP and 8-NH2-dGTP by reverse transcriptases and mammalian DNA polymerases, J. Biol. Chem. 272 (1997) 5892–5898. [3] A. Ramanathan, L. Pape, D.C. Schwartz, High-density polymerase-mediated incorporation of fluorochrome-labeled nucleotides, Anal. Biochem. 337 (2005) 1–11. [4] M.M. Butler, G.E. Wright, A method to assay inhibitors of DNA polymerase IIIC activity, in: W.S. Champney (Ed.), Methods in Molecular Medicine: New Antibiotic Targets, Humana Press, Totowa, NJ, 2008, pp. 25–36. [5] J.R. Brody, S.E. Kern, History and principles of conductive media for standard DNA electrophoresis, Anal. Biochem. 333 (2004) 1–13. [6] J.R. Brody, S.E. Kern, Sodium boric acid: A Tris-free, Cooler conductive medium for DNA electrophoresis, BioTechniques 36 (2004) 214–216. [7] S.S. Negro, R. Cloots, N.J. Gemmell, Cost-effective media for the rapid and high resolution of small DNA fragments using polyacrylamide-based electrophoresis, Mol. Ecol. Notes 6 (2006) 609–612. [8] Y. Mizushina, N. Tanaka, H. Yagi, T. Kurosawa, M. Onoue, H. Seto, T. Horie, N. Aoyagi, M. Yamaoka, A. Matsukage, S. Yoshida, K. Sakaguchi, Fatty acids selectively inhibit eukaryotic DNA polymerase activities in vitro, Biochem. Biophys. Acta 1308 (1996) 256–262. [9] J. Sambrook, D.W. Russell (Eds.), Molecular Cloning: A Laboratory Manual, third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001, p. 12.108 (for taurine) and p. 12.75 (for TBE).
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Rapid sequencing gel electrophoresis using glycerol-tolerant sodium taurine medium Ken Hirano *, Tomomi Ishido, Mitsuru Ishikawa Health Technology Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu, Kagawa 761-0395, Japan
a r t i c l e
i n f o
Article history: Received 23 March 2009 Available online 11 April 2009
a b s t r a c t We describe a new glycerol-tolerant sodium taurine (ST) medium for rapid sequencing gel electrophoresis by substituting the standard conductive media of Tris–boric acid–ethylenediaminetetraacetic acid (EDTA) (TBE) and Tris–taurine–EDTA (TTE) and other low-ionic-strength media of sodium boric acid (SB). Low-ionic-strength and cost-effective ST media gave glycerol tolerance up to 50% (v/v) glycerol-containing DNA sample solution, shorter running time, and better resolution to separate small DNA oligonucleotides (20–45 mer) in 12% denaturing sequencing gel electrophoresis. Ó 2009 Elsevier Inc. All rights reserved.
Sequencing gel electrophoresis is an essential tool to analyze not only DNA sequences but also DNA polymerase for characterization of new recombinant enzymes [1], the incorporation activity of 20 -deoxyribonucleoside 50 -triphosphate (dNTP)1 derivatives for mutagenic effects and single-molecule sequencing [2,3], and inhibitor assays for molecular medicine [4]. The standard conducting media of Tris–boric acid–ethylenediaminetetraacetic acid (EDTA) (TBE) and Tris–taurine–EDTA (TTE) used for sequencing gel electrophoresis have a disadvantage in that a high voltage cannot be applied due to the high ionic strength of the media [5]. A long running time of gel electrophoresis is required, therefore, and is achieved by using standard media and a long-sized sequencing gel. A novel low-ionicstrength sodium boric acid (SB) medium was recently shown to shorten the running time and improve the resolution of DNA fragments in not only an agarose gel system [6] but also a sequencing gel system to analyze small DNA fragments (90–300 bp) [7]. In the current study, we found that SB medium was affected by glycerol in the reaction sample. Some DNA polymerases required a high concentration of glycerol in the reaction buffer (e.g., mammalian DNA polymerase-b required reaction buffer containing 15% [v/v] glycerol) [8]. Direct sample loading to the gel was needed for quantitative primer extension assay without DNA purification for DNA polymerase requiring a high concentration of glycerol and for commercial DNA polymerases stored in buffer containing 50% (v/v) glycerol. In the current study, we used new low-ionic-strength sodium taurine (ST) medium and evaluated glycerol-tolerant conducting media by
* Corresponding author. Fax: +81 87 869 3569. E-mail address: [email protected] (K. Hirano). 1 Abbreviations used: dNTP, 20 -deoxyribonucleoside 50 -triphosphate; EDTA, ethylenediaminetetraacetic acid; TBE, Tris–boric acid–EDTA; TTE, Tris–taurine–EDTA; SB, sodium boric acid; ST, sodium taurine; DIG, digoxygenin; PAGE, polyacrylamide gel electrophoresis; APS, ammonium persulfate. 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.04.006
substituting SB media for ST media for rapid sequencing gel electrophoresis in the presence of glycerol in the loading samples. The sequencing gel electrophoresis equipment used was the Sequi-Gen GT system (Bio-Rad, Hercules, CA, USA). As a glycerol-tolerant medium, ST medium was prepared at the final concentrations of 5 mM sodium hydroxide and 11 mM taurine (first grade, >98.5%, Sigma–Aldrich Japan, Tokyo, Japan) at pH 9.0. Comparison media of SB (10 mM sodium hydroxide [pH 8.5] adjusted with boric acid) [6], TBE (89 mM Tris, 89 mM boric acid, and 2 mM EDTA) [9], and TTE (89 mM Tris, 29 mM taurine [2-aminoethanesulfonic acid], and 0.1 mM EDTA) [9] were used. Digoxygenin (DIG)-labeled oligonucleotides (20–45 mer) were synthesized and purchased from Sigma–Aldrich Japan and analyzed on 12% denaturing polyacrylamide gel. Gels were 0.4 mm thick and 50 cm long. Different concentrations of glycerol (0–50%, v/v) in the DNA sample solution were tested in each conductive medium. Power during the electrophoresis was fixed at 50 W. Voltage, current, and temperature were recorded at the start and end of electrophoresis. After electrophoresis, separated DNA fragments were detected by chemiluminescence with a DIG detection system [2]. Chemiluminescent images were captured using X-ray film. Fig. 1 shows the separation of small oligonucleotides (20– 45 mer) using four conductive media (ST, SB, TTE, and TBE) in 12% denaturing sequencing gel electrophoresis at different glycerol concentrations (0–50%, v/v). A range of excess glycerol concentrations up to 50% (v/v) was selected to indicate the effect of glycerol. ST and TTE of taurine-based media gave glycerol-tolerant separation up to 50% (v/v) glycerol in high concentration. SB and TBE of boric acid-based media were affected by band blurring that was dependent on the glycerol concentration in the sample solution. In SB medium, bending in the sequencing tracks and shrinkage of the width of bands were observed, resulting in desirable resolution in the presence of glycerol in sequencing gel electrophoresis. The effect of glycerol in the boric acid-based
Notes & Tips / Anal. Biochem. 390 (2009) 100–101
101
Fig. 1. Analysis of small DNA fragments containing glycerol in sample solutions using various conductive media in 12% denaturing polyacrylamide sequencing gel electrophoresis. The DNA sample solution was a mixture of oligonucleotides of 20, 21, 22, 23, 24, 25, 30, 35, 40, and 45 mer. The concentrations of glycerol in DNA sample solutions were 0, 5, 10, 20, 30, 40, and 50% (v/v). Running time was established when the distance between 20 and 45 mer for the 0% glycerol sample was 11 cm for each conductive medium. Power during the electrophoresis was fixed at 50 W. Voltage, current, and temperature were recorded at the start and end of gel electrophoresis.
medium was due to the interaction of glycerol with boric acid in conductive media to form anionic esters, which migrate through the gel during electrophoresis [9]. When replacing boric acid in the gel and in the electrophoresis buffer with taurine, the latter suppressed interaction of glycerol in conductive media because of a weak aminosulfonic acid with a pKa of 9.06, similar to the pKa of boric acid (9.24) [9]. ST, as a taurine-based medium similar to TTE medium, suppressed the effect of glycerol interaction at the extreme end of glycerol concentration in the current study. ST medium gave not only glycerol tolerance but also rapid separation. The low ion conduction of ST medium also enabled the application of high voltage (by Ohm’s law) at a constant power of 50 W during gel electrophoresis. Application of high voltage reduced the running time of gel electrophoresis by 26% (119 min instead of 160 min) per run. The effect of this reduction may be important in sequencing gel electrophoresis because a long run time is necessary. The difference in temperature rise between each medium was small because the current used during electrophoresis between each medium was not greatly different (Fig. 1). The current in polyacrylamide gel electrophoresis (PAGE) is derived from not only the conducting media but also ammonium persulfate (APS) in the gel. This is different from agarose gel electrophoresis. APS, being a salt, may have affected the current to some extent and seems to affect the current difference in temperature rise. ST medium and SB medium are cost-effective; their reagent costs are 10-fold less expensive compared with Tris-based standard media [5]. In conclusion, we found that ST medium was glycerol tolerant and gave rapid separation of DNA fragments in sequencing gel electrophoresis. Glycerol-tolerant ST medium may also enable high-throughput analysis because of not only rapid separation but also labor-free sample loading on the sequencing gel without previous purification of reaction samples to remove glycerol. The
current study is important for developing a method for glyceroltolerant rapid analysis of DNA and RNA fragments using not only standard sequencing gel electrophoresis and native PAGE (which have been used for single-strand conformation polymorphisms) but also capillary and microfluidic chip-based electrophoresis for high-throughput analysis. Acknowledgment This work was supported by a grant from the Industrial Technology Research Program from the New Energy and Industrial Technology Development Organization (NEDO), Japan. References [1] N. Shimazaki, K. Yoshida, T. Kobayashi, S. Toji, K. Tamai, O. Koiwai, Overexpression of human DNA polymerase lambda in E. Coli and characterization of the recombinant enzyme, Gene Cells 7 (2002) 639–651. [2] A.S. Kamath-Loeb, A. Hizi, H. Kasai, L.A. Loeb, Incorporation of the guanosine triphosphate analog 8-oxo-dGTP and 8-NH2-dGTP by reverse transcriptases and mammalian DNA polymerases, J. Biol. Chem. 272 (1997) 5892–5898. [3] A. Ramanathan, L. Pape, D.C. Schwartz, High-density polymerase-mediated incorporation of fluorochrome-labeled nucleotides, Anal. Biochem. 337 (2005) 1–11. [4] M.M. Butler, G.E. Wright, A method to assay inhibitors of DNA polymerase IIIC activity, in: W.S. Champney (Ed.), Methods in Molecular Medicine: New Antibiotic Targets, Humana Press, Totowa, NJ, 2008, pp. 25–36. [5] J.R. Brody, S.E. Kern, History and principles of conductive media for standard DNA electrophoresis, Anal. Biochem. 333 (2004) 1–13. [6] J.R. Brody, S.E. Kern, Sodium boric acid: A Tris-free, Cooler conductive medium for DNA electrophoresis, BioTechniques 36 (2004) 214–216. [7] S.S. Negro, R. Cloots, N.J. Gemmell, Cost-effective media for the rapid and high resolution of small DNA fragments using polyacrylamide-based electrophoresis, Mol. Ecol. Notes 6 (2006) 609–612. [8] Y. Mizushina, N. Tanaka, H. Yagi, T. Kurosawa, M. Onoue, H. Seto, T. Horie, N. Aoyagi, M. Yamaoka, A. Matsukage, S. Yoshida, K. Sakaguchi, Fatty acids selectively inhibit eukaryotic DNA polymerase activities in vitro, Biochem. Biophys. Acta 1308 (1996) 256–262. [9] J. Sambrook, D.W. Russell (Eds.), Molecular Cloning: A Laboratory Manual, third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001, p. 12.108 (for taurine) and p. 12.75 (for TBE).