Chromatographia

, Volume 76, Issue 7–8, pp 329–334 | Cite as

Isolation of Xis Gen Fragment of λ Phage from Agarose Gel Using Magnetic Particles for Subsequent Enzymatic DNA Sequencing

  • Kristyna Smerkova
  • Simona Dostalova
  • Helena Skutkova
  • Marketa Ryvolova
  • Vojtech Adam
  • Ivo Provaznik
  • Rene Kizek
Original
First Online:
Received:
Revised:
Accepted:

Abstract

Gel electrophoresis is one of the most important methods used in biochemistry and molecular biology. The recovery of analytes from the gel required for subsequent analysis including amplification by polymerase chain reaction (PCR) or DNA sequencing is an issue due to the gel contamination. Among the other methods used for sample recovery, the application of nanomaterials is also being investigated. In this study, the applicability of magnetic particles (1 μm) for isolation of DNA fragment from agarose gel with subsequent DNA sequencing was investigated. Electrochemical analysis and DNA sequencing was used to investigate the recovery yield. The influence of dilution of the gel prior to the purification was investigated and the linear dependence with regression coefficient R 2 = 0.9972 was obtained using square wave voltammetry. Moreover, bioinformatic analysis was used for comparison of obtained sequences, and simple and easy identification of non-systematic errors caused by both fluorescence labeling reaction and electrophoretic separation. It was found that magnetic nanoparticles based isolation markedly lowered the errors occurring during sequencing of the isolated DNA fragment from 7 to 1 %.

Keywords

Gel electrophoresis Electrochemical analysis DNA sequencing DNA purification Magnetic particles 
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Notes

Acknowledgments

Financial support from the following projects IGA IP 19/2012, NanoBioTECell GACR P102/11/1068 and CEITEC CZ.1.05/1.1.00/02.0068 is highly acknowledged. The author M.R. wishes to express her thanks to project CZ.1.07/2.3.00/30.039 for financial support.

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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Kristyna Smerkova
    • 1
  • Simona Dostalova
    • 1
    • 2
  • Helena Skutkova
    • 2
  • Marketa Ryvolova
    • 1
    • 3
  • Vojtech Adam
    • 1
    • 3
  • Ivo Provaznik
    • 2
  • Rene Kizek
    • 1
    • 3
  1. 1.Department of Chemistry and Biochemistry, Faculty of AgronomyMendel University in BrnoBrnoCzech Republic
  2. 2.Department of Biomedical Engineering, Faculty of Electrical Engineering and CommunicationBrno University of TechnologyBrnoCzech Republic
  3. 3.Central European Institute of TechnologyBrno University of TechnologyBrnoCzech Republic

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