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An Improved Method of Bisulfite Treatment and Purification to Study Precise DNA Methylation from as Little as 10 pg DNA

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Abstract

Methylation of vertebrate DNA is one of the most important epigenetic alterations which have become a center of scientific attraction, especially because of its important role in the regulation of transcription, genomic imprinting, developmental process, and pathogenesis of various diseases. Currently, there are wide ranges of methods available to produce quantitative and qualitative information on genomic DNA methylation. The vast majority of these methods rely on the optimization of the efficient bisulfite treatment. However, all the available methods for bisulfite treatment suffer from major disadvantages, such as large amount of starting material, poor conversion efficiency as well as low recovery and integrity of DNA after bisulfite treatment. Here, we developed a simple, rapid, and convenient column-based bisulfite treatment method by improving the several critical steps, which leads to consistent C-to-U conversion rate 99–100 %, >75 % recovery of DNA after bisulfite treatment. In addition, it is commercially viable and requires very less amount (∼10 pg) of DNA.

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Acknowledgments

This work was supported under the World Bank funded NAIP Project C 30015 of Indian Council of Agriculture Research, New Delhi, India.

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Authors and Affiliations

  1. Animal Genomics Lab, ABTC, NDRI, Karnal, 132001, India

    Sandeep K. Rajput, Sandeep Kumar, Ankita Rajput & Tirtha K. Datta

  2. Amity Institute of Biotechnology, Amity University, Noida, Uttar Pradesh, India

    Vivek P. Dave

  3. Department of Biochemistry, Banaras Hindu University, Varanasi, India

    Haushila P. Pandey

  4. Genomics Lab, Animal Biotechnology Centre, National Dairy Research Institute, Karnal, 132001, Haryana, India

    Tirtha K. Datta

Authors
  1. Sandeep K. Rajput
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  2. Sandeep Kumar
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  3. Vivek P. Dave
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  4. Ankita Rajput
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  5. Haushila P. Pandey
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  6. Tirtha K. Datta
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Corresponding author

Correspondence to Tirtha K. Datta.

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Rajput, S.K., Kumar, S., Dave, V.P. et al. An Improved Method of Bisulfite Treatment and Purification to Study Precise DNA Methylation from as Little as 10 pg DNA. Appl Biochem Biotechnol 168, 797–804 (2012). https://doi.org/10.1007/s12010-012-9820-7

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  • DOI: https://doi.org/10.1007/s12010-012-9820-7

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