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Protocol for the quantitative assessment of DNA concentration and damage (fragmentation and nicks)

Nature Protocols volume 4, pages 125–131 (2009)Cite this article

Abstract

An ultrasensitive protocol is presented for the quantitative assessment of fragmented and nicked dsDNA using PicoGreen and consists of four methods. The first quantifies the concentration of DNA, whereas the second (quantitative complement of the Comet assay) quantifies the degree of DNA fragmentation seen in a typical DNA agarose electrophoresis gel. Both methods have sensitivity of 5 pg of DNA. The third method (quantitative counterpart of the electrophoresis-based qualitative apoptotic and necrotic DNA assays) quantifies the polyethylene glycol-fractionated small-size (0–1 kb) fragmented DNA. This method also detects up to 5 pg of damaged DNA and requires a minimum sample of quantity 0.2 ml of 2.5 μg ml−1. The fourth method measures the percentage of DNA nicks by alkaline DNA unwinding and requires up to 15 pg of DNA sample. The time required for processing 10 DNA samples is 1/2, 1, 13 and 1 h for the first, second, third and fourth method, respectively.

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Figure 1: Fluorescence dependence of PicoGreen–DNA complex on the degree of DNA fragmentation13.
Figure 2: Quantitative assessment of DNA fragmentation smearing in agarose gel electrophoresis by this protocol13.
Figure 3: Principle of small-size (0–1 kb) fragmented DNA quantification14.
Figure 4: Quantification of DNA small-size fragmentation and nicks in filamentous phytopathogenic fungi.

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Acknowledgements

This work was financially supported by the Greek Ministry of Education, University of Patras, Greece.

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Authors and Affiliations

  1. Department of Biology, Section of Genetics, Cell Biology and Development, University of Patras, 26100—Patras, Greece.,

    Christos D Georgiou, Ioannis Papapostolou & Konstantinos Grintzalis

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  1. Christos D Georgiou
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  2. Ioannis Papapostolou
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  3. Konstantinos Grintzalis
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Correspondence to Christos D Georgiou.

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Georgiou, C., Papapostolou, I. & Grintzalis, K. Protocol for the quantitative assessment of DNA concentration and damage (fragmentation and nicks). Nat Protoc 4, 125–131 (2009). https://doi.org/10.1038/nprot.2008.222

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