Skip to main content
SpringerLink
Log in

Miniaturized Sequencing Gel System for Quick Analysis of DNA by Hydroxyl Radical Cleavage

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

We described a simple and quick miniaturized sequencing gel system for DNA analysis. Two major modifications were made to the previously reported miniaturized DNA sequencing gel system to achieve high-resolution hydroxyl radical cleavage analysis: including formamide in the miniaturized gel and providing uniform heating during electrophoresis. Our method enables one to reduce the cost for chemicals and to significantly reduce electrophoresis time. Furthermore, minimal gel handling simplifies the entire process. We show that the resolution of DNA fragments obtained by hydroxyl radical cleavage for the miniaturized gel is similar to that of a large conventional sequencing gel.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Greenbaum, J. A., Pang, B., & Tullius, T. D. (2007). Construction of a genome-scale structural map at single-nucleotide resolution. Genome Research, 17, 947–953.

    Article  CAS  Google Scholar 

  2. Tullius, T. D., & Dombroski, B. A. (1985). Iron(II) EDTA used to measure the helical twist along any DNA molecule. Science, 230, 679–681.

    Article  CAS  Google Scholar 

  3. Takasuka, T. E., & Stein, A. (2010). Direct measurements of the nucleosome-forming preferences of periodic DNA motifs challenge established models. Nucleic Acids Research, 38, 5672–5680.

    Article  CAS  Google Scholar 

  4. Bishop, E. P., Rohs, R., Parker, S. C., West, S. M., Liu, P., Mann, R. S., Honig, B., & Tullius, T. D. (2011). A map of minor groove shape and electrostatic potential from hydroxyl radical cleavage patterns of DNA. ACS Chemical Biology, 6, 1314–1320.

    Article  CAS  Google Scholar 

  5. Margolin, Y., Shafirovich, V., Geacintov, N. E., DeMott, M. S., & Dedon, P. C. (2008). DNA sequence context as a determinant of the quantity and chemistry of guanine oxidation produced by hydroxyl radicals and one-electron oxidants. Journal of Biological Chemistry, 283, 35569–35578.

    Article  CAS  Google Scholar 

  6. Takasuka, T. E., Cioffi, A., & Stein, A. (2008). Sequence information encoded in DNA that may influence long-range chromatin structure correlates with human chromosome functions. PLoS One, 3, e2643.

    Article  Google Scholar 

  7. Jain, S. S., & Tullius, T. D. (2008). Footprinting protein–DNA complexes using the hydroxyl radical. Nature Protocols, 3, 1092–1100.

    Article  CAS  Google Scholar 

  8. Stein, A., Hill, S. A., Cheng, Z., & Bina, M. (1998). Simple miniaturized gel system for DNA sequence analysis. Nucleic Acids Research, 26, 452–455.

    Article  CAS  Google Scholar 

  9. Maxam, A. M., & Gilbert, W. (1980). Sequencing end-labeled DNA with base-specific chemical cleavages. Methods in Enzymology, 65, 499–560.

    Article  CAS  Google Scholar 

  10. Blake, R. D., & Delcourt, S. G. (1996). Thermodynamic effects of formamide on DNA stability. Nucleic Acids Research, 24, 2095–2103.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors thank the Department of Biological Sciences, Purdue University, West Lafayette.

Author information

Authors and Affiliations

  1. Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA

    Taichi E. Takasuka

  2. Department of Urology, Stanford University, Stanford, CA, USA

    Yi-Ju Hsieh

  3. Department of Biological Sciences, Purdue University, West Lafayette, IN, USA

    Taichi E. Takasuka, Yi-Ju Hsieh & Arnold Stein

Authors
  1. Taichi E. Takasuka
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yi-Ju Hsieh
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Arnold Stein
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Taichi E. Takasuka.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Takasuka, T.E., Hsieh, YJ. & Stein, A. Miniaturized Sequencing Gel System for Quick Analysis of DNA by Hydroxyl Radical Cleavage. Appl Biochem Biotechnol 172, 1–8 (2014). https://doi.org/10.1007/s12010-013-0512-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-013-0512-8

Keywords

Search

Navigation