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Whole-genome fractionation rapidly purifies DNA from centromeric regions

Nature Methods volume 1, pages 67–71 (2004)Cite this article

Abstract

The condensed centromeric regions of higher eukaryotic chromosomes contain satellite sequences, transposons and retroelements, as well as transcribed genes that perform a variety of functions. These chromosomal domains nucleate kinetochores, mediate sister chromatid cohesion and inhibit recombination, yet their characterization has often lagged behind that of chromosome arms. Here, we describe a whole-genome fractionation technique that rapidly identifies bacterial artificial chromosome (BAC) clones derived from plant centromeric regions. This approach, which relies on hybridization of methylated genomic DNA, revealed BACs that correspond to the genetically mapped and sequenced Arabidopsis thaliana centromeric regions. Extending this method to other species in the Brassicaceae family identified centromere-linked clones and provided genome-wide estimates of methylated DNA abundance. Sequencing these clones will elucidate the changes that occur during plant centromere evolution. This genomic fractionation technique could identify centromeric DNA in genomes with similar methylation and repetitive DNA content, including those from crops and mammals.

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Figure 1: Whole-genome fractionation of A. thaliana.
Figure 2: Abundance and location of BACs identified by whole-genome fractionation.
Figure 3: Localization of C. rubella class I BACs.

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Acknowledgements

This work was supported by a US National Science Foundation postdoctoral fellowship in bioinformatics (A.E.H.) and grants from the National Science Foundation, Atlantic Philanthropies and Howard Hughes Medical Institute. We thank E. Bray, K.C. Keith and R. Palanivelu for critical manuscript reading, G. Kettler for computational analyses and J. Mach for assistance with FISH protocols.

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Authors and Affiliations

  1. Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, 60637, Illinois, USA

    Song Luo, Anne E Hall, Sarah E Hall & Daphne Preuss

  2. Department of Molecular Genetics and Cell Biology, Howard Hughes Medical Institute, University of Chicago, Chicago, 60637, Illinois, USA

    Song Luo & Daphne Preuss

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  1. Song Luo
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Correspondence to Daphne Preuss.

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Competing interests

S.L. and D.P. hold shares in the company Chromatin Inc., which is constructing autonomous chromosomes in plants as vehicles for delivering commercially important traits to crops..

Supplementary information

Supplementary Table 1

Arabidopsis thaliana Class I BACs. (PDF 24 kb)

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Luo, S., Hall, A., Hall, S. et al. Whole-genome fractionation rapidly purifies DNA from centromeric regions. Nat Methods 1, 67–71 (2004). https://doi.org/10.1038/nmeth703

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