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Preparation of megabase-sized DNA from a variety of organisms using the nuclei method for advanced genomics research

Nature Protocols volume 7, pages 467–478 (2012)Cite this article

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Abstract

Megabase-sized DNA is crucial to modern genomics research of all organisms. Among the preparation methods developed, the nuclei method is the simplest and most widely used for preparing high-quality megabase-sized DNA from divergent organisms. In this method, nuclei are first isolated by physically grinding the source tissues. The nontarget cytoplast organellar genomes and metabolites are removed by centrifugation and washing, thus maximizing the utility of the method and substantially improving the digestibility and clonability of the resultant DNA. The nuclei are then embedded in an agarose matrix containing numerous pores, allowing the access of restriction enzymes while preventing the DNA from physical shearing. DNA is extracted from the nuclei, purified and subsequently manipulated in the agarose matrix. Here we describe the nuclei method that we have successfully used to prepare high-quality megabase-sized DNA from hundreds of plant, animal, fish, insect, algal and microbial species. The entire protocol takes ∼3 d.

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Figure 1: An overview of the methods for megabase-sized DNA isolation from cells, protoplasts or nuclei.
Figure 2: Megabase-sized DNA isolated using the protocols presented in this article.

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Acknowledgements

We thank the former members of H.-B.Z.'s laboratory who have used the protocols described in this article for their research and thus might have directly or indirectly contributed to the protocol. This work is supported in part by Research Grant Award no. IS-4427-11C from BARD, the United States-Israel Binational Agricultural Research and Development Fund (H.-B.Z.) and grants (124475-85360 and 124329-85360) from the Texas AgriLife Research Cotton and Monocot Crop Improvement Program (H.-B.Z.).

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Author notes

  1. Cheng-Cang Wu & Jennifer J Dong

    Present address: Present addresses: Lucigen Corporation, Middleton, Wisconsin, USA (C.-C.W.) and Department of Microbial and Molecular Pathogenesis, Texas A&M Health Science Center, College of Medicine, College Station, Texas, USA (J.J.D.).,

Authors and Affiliations

  1. Department of Soil and Crop Sciences, Texas A&M University, College Station, Texas, USA

    Meiping Zhang, Yang Zhang, Chantel F Scheuring, Cheng-Cang Wu, Jennifer J Dong & Hong-Bin Zhang

  2. College of Life Science, Jilin Agricultural University, Changchun, Jilin, China

    Meiping Zhang

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Contributions

M.P.Z., Y.Z., C.F.S., C.-C.W., J.J.D. and H.-B.Z. conducted the experiments on different species with the protocol presented here, and improved and extended the original protocol through these experiments. M.P.Z. wrote the manuscript; H.-B.Z. developed the original concept of the protocol and the original protocol, designed the experiments and wrote the manuscript.

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Correspondence to Hong-Bin Zhang.

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Zhang, M., Zhang, Y., Scheuring, C. et al. Preparation of megabase-sized DNA from a variety of organisms using the nuclei method for advanced genomics research. Nat Protoc 7, 467–478 (2012). https://doi.org/10.1038/nprot.2011.455

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