Advertisement

Nano Research

, Volume 8, Issue 8, pp 2714–2720 | Cite as

A green method of staining DNA in polyacrylamide gel electrophoresis based on fluorescent copper nanoclusters synthesized in situ

  • Xiaoli Zhu
  • Hai Shi
  • Yalan Shen
  • Bin Zhang
  • Jing Zhao
  • Genxi LiEmail author
Research Article

Abstract

The safety of nucleic acid staining dyes has long been recognized to be a problem. Extensive efforts have been made to search for alternatives to the most popular but toxic staining dye, ethidium bromide (EtBr). However, so far no staining method that can be guaranteed to be sufficiently safe has been developed. In this paper, we report a green staining method of DNA in polyacrylamide gel electrophoresis, where in situ synthesis of DNA-templated fluorescent copper nanoclusters (CuNCs) in the gel is achieved to make the DNA bands visible under UV light. Moreover, a comprehensive study of the performance of this staining method has been conducted and the experimental results show that it has favorable sensitivity, stability, and usability. Meanwhile, in our animal experiments, the two reagents (copper sulfate and ascorbic acid) as well as the synthesized CuNCs have been proven to be non-toxic in contact with skin. In addition, all the reagents employed in this work are readily available and low cost, and the procedure is simple to carry out. Therefore, this novel staining method based on the in situ synthesis DNA-templated fluorescent CuNCs has many potential applications.

Keywords

staining nucleic acids polyacrylamide gel copper nanoclusters electrophoresis 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Supplementary material

12274_2015_778_MOESM1_ESM.pdf (2 mb)
Supplementary material, approximately 1.95 MB.

References

  1. [1]
    Meyers, J. A.; Sanchez, D.; Elwell, L. P.; Falkow, S. Simple agarose gel electrophoretic method for the identification and characterization of plasmid deoxyribonucleic acid. J. Bacteriol. 1976, 127, 1529–1537.Google Scholar
  2. [2]
    Lee, J. D.; Huang, C. H.; Wang, N. W.; Lu, C. S. Automatic DNA sequencing for electrophoresis gels using image processing algorithms. J. Biomed. Sci. Eng. 2011, 4, 523–528.CrossRefGoogle Scholar
  3. [3]
    Ogier, J. C.; Son, O.; Gruss, A.; Tailliez, P.; Delacroix-Buchet, A. Identification of the bacterial microflora in dairy products by temporal temperature gradient gel electrophoresis. Appl. Environ. Microb. 2002, 68, 3691–3701.CrossRefGoogle Scholar
  4. [4]
    Rotaru, A.; Dutta, S.; Jentzsch, E.; Gothelf, K.; Mokhir, A. Selective dsDNA-templated formation of copper nanoparticles in solution. Angew. Chem. Int. Edit. 2010, 49, 5665–5667.CrossRefGoogle Scholar
  5. [5]
    Yi, S. H.; Xu, L. C.; Mei, K.; Yang, R. Z.; Huang, D. X. Isolation and identification of age-related DNA methylation markers for forensic age-prediction. Forensic. Sci. Int.: Genet. 2014, 11, 117–125.CrossRefGoogle Scholar
  6. [6]
    Aaij, C.; Borst, P. The gel electrophoresis of DNA. Biochim. Biophys. Acta, Nucleic Acids Protein Synth. 1972, 269, 192–200.CrossRefGoogle Scholar
  7. [7]
    Singer, V. L.; Lawlor, T. E.; Yue, S. Comparison of SYBR (R) green I nucleic acid gel stain mutagenicity and ethidium bromide mutagenicity in the salmonella/mammalian microsome reverse mutation assay (ames test). Mutat. Res., Genet. Toxicol. Environ. Mutagen. 1999, 439, 37–47.CrossRefGoogle Scholar
  8. [8]
    Ohta, T.; Tokishita, S.; Yamagata, H. Ethidium bromide and SYBR Green I enhance the genotoxicity of UV-irradiation and chemical mutagens in E-coli. Mutat. Res., Genet. Toxicol. Environ. Mutagen. 2001, 492, 91–97.CrossRefGoogle Scholar
  9. [9]
    Yang, Y. Y.; Chen, S. W. Surface manipulation of the electronic energy of subnanometer-sized gold clusters: An electrochemical and spectroscopic investigation. Nano. Lett. 2003, 3, 75–79.CrossRefGoogle Scholar
  10. [10]
    Peyser, L. A.; Vinson, A. E.; Bartko, A. P.; Dickson, R. M. Photoactivated fluorescence from individual silver nanoclusters. Science. 2001, 291, 103–106.CrossRefGoogle Scholar
  11. [11]
    Hicks, J. F.; Miles, D. T.; Murray, R. W. Quantized doublelayer charging of highly monodisperse metal nanoparticles. J. Am. Chem. Soc. 2002, 124, 13322–13328.CrossRefGoogle Scholar
  12. [12]
    Vosch, T.; Antoku, Y.; Hsiang, J. C.; Richards, C. I.; Gonzalez, J. I.; Dickson, R. M. Strongly emissive individual DNAencapsulated Ag nanoclusters as single-molecule fluorophores. Proc. Natl. Acad. Sci. USA 2007, 104, 12616–12621.CrossRefGoogle Scholar
  13. [13]
    Huang, C. C.; Yang, Z.; Lee, K. H.; Chang, H. T. Synthesis of highly fluorescent gold nanoparticles for sensing Mercury(II). Angew. Chem. Int. Edit. 2007, 46, 6824–6828.CrossRefGoogle Scholar
  14. [14]
    Seeman, N. C. DNA in a material world. Nature 2003, 421, 427–431.CrossRefGoogle Scholar
  15. [15]
    Lin, C. X.; Liu, Y.; Rinker, S.; Yan, H. DNA tile based self-assembly: Building complex nanoarchitectures. ChemPhysChem 2006, 7, 1641–1647.CrossRefGoogle Scholar
  16. [16]
    Lin, C. X.; Liu, Y.; Yan, H. Designer DNA nanoarchitectures. Biochemistry 2009, 48, 1663–1674.CrossRefGoogle Scholar
  17. [17]
    Sharma, J.; Yeh, H. C.; Yoo, H.; Werner, J. H.; Martinez, J. S. A complementary palette of fluorescent silver nanoclusters. Chem. Commun. 2010, 46, 3280–3282.CrossRefGoogle Scholar
  18. [18]
    Rotaru, A.; Dutta, S.; Jentzch, E.; Gothelf, K.; Mokhir, A. Selective dsDNA-templated formation of copper nanoparticles in solution. Angew. Chem. Int. Ed. 2010, 49, 5665–5667.CrossRefGoogle Scholar
  19. [19]
    Petty, J. T.; Zheng, J.; Hud, N. V.; Dickson, R. M. DNAtemplated Ag nanocluster formation. J. Am. Chem. Soc. 2004, 126, 5207–5212.CrossRefGoogle Scholar
  20. [20]
    Liu, G. Y.; Shao, Y.; Ma, K.; Cui, Q. H.; Wu, F.; Xu, S. J. Synthesis of DNA-templated fluorescent gold nanoclusters. Gold Bull. 2012, 45, 69–74.CrossRefGoogle Scholar
  21. [21]
    Monson, C. F.; Woolley, A. T. DNA-templated construction of copper nanowires. Nano lett. 2003, 3, 359–363.CrossRefGoogle Scholar
  22. [22]
    Qing, Z. H.; He, X. X.; He, D. G.; Wang, K. M.; Xu, F. Z.; Qing, T. P.; Yang, X. Poly(thymine)-templated selective formation of fluorescent copper nanoparticles. Angew. Chem. Int. Edit. 2013, 52, 9719–9722.CrossRefGoogle Scholar
  23. [23]
    Liu, G. Y.; Shao, Y.; Peng, J.; Dai, W.; Liu, L. L.; Xu, S. J.; Wu, F.; Wu, X. H. Highly thymine-dependent formation of fluorescent copper nanoparticles templated by ss-DNA. Nanotechnology 2013, 24, 345502.CrossRefGoogle Scholar
  24. [24]
    Zhang, L. L.; Zhao, J. J.; Duan, M.; Zhang, H.; Jiang, J. H.; Yu, R. Inhibition of dsDNA-templated copper nanoparticles by pyrophosphate as a label-free fluorescent strategy for alkaline phosphatase assay. Anal. Chem. 2013, 85, 3797–3801.CrossRefGoogle Scholar
  25. [25]
    Xu, F. Z.; Shi, H.; He, X. X.; Wang, K.; He, D. G.; Guo, Q.; Qing, Z. H.; Yan, L. A.; Ye, X. S.; Li, D. et al. Concatemeric dsDNA-templated copper nanoparticles strategy with improved sensitivity and stability based on rolling circle replication and its application in microRNA detection. Anal. Chem. 2014, 86, 6976–6982.CrossRefGoogle Scholar
  26. [26]
    Chen, J. H.; Liu, J.; Fang, Z. Y.; Zeng, L. W. Random dsDNA-templated formation of copper nanoparticles as novel fluorescence probes for label-free lead ions detection. Chem. Commun. 2012, 48, 1057–1059.CrossRefGoogle Scholar
  27. [27]
    Pacioni, N. L.; Filippenko, V.; Presseau, N.; Scaiano, J. C. Oxidation of copper nanoparticles in water: Mechanistic insights revealed by oxygen uptake and spectroscopic methods. Dalton Trans. 2013, 42, 5832–5838.CrossRefGoogle Scholar
  28. [28]
    Chen, T. S.; Zeng, S. Q.; Zhou, W.; Luo, Q. M. A. Quantitative theory model of a photobleaching mechanism. Chinese Phys. Lett. 2003, 20, 1940–1943.CrossRefGoogle Scholar

Copyright information

© Tsinghua University Press and Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Xiaoli Zhu
    • 1
  • Hai Shi
    • 1
  • Yalan Shen
    • 1
  • Bin Zhang
    • 1
  • Jing Zhao
    • 1
  • Genxi Li
    • 1
    • 2
    Email author
  1. 1.Laboratory of Biosensing Technology, School of Life SciencesShanghai UniversityShanghaiChina
  2. 2.State Key Laboratory of Pharmaceutical Biotechnology, Department of BiochemistryNanjing UniversityNanjingChina

Personalised recommendations