Abstract
DNaseI-hypersensitive sites within chromatin are indicative of genomic loci with regulatory function. Several techniques have been described for analyzing these regions, but are either laborious, offer low-throughput possibilities, or are expensive. We have developed a new approach based on a modified version of multiplex ligation-dependent probe amplification (MLPA). Using this method, it is possible to analyse up to 50 defined genomic regions for DNaseI-hypersensitivity in a single PCR-based reaction. This chapter outlines the approach and discusses the critical features of each step of the procedure.
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References
Gross, D. S., and Garrard, W. T. (1988) Nuclease hypersensitive sites in chromatin. Annual review of biochemistry 57, 159–197.
Wu, C., Wong, Y. C., and Elgin, S. C. (1979) The chromatin structure of specific genes: II. Disruption of chromatin structure during gene activity. Cell 16, 807–814.
Burgess-Beusse, B., Farrell, C., Gaszner, M., Litt, M., Mutskov, V., Recillas-Targa, F., Simpson, M., West, A., and Felsenfeld, G. (2002) The insulation of genes from external enhancers and silencing chromatin. Proceedings of the National Academy of Sciences of the United States of America 99 Suppl 4, 16433–16437.
Follows, G. A., Janes, M. E., Vallier, L., Green, A. R., and Gottgens, B. (2007) Real-time PCR mapping of DNaseI-hypersensitive sites using a novel ligation-mediated amplification technique. Nucleic acids research 35, e56.
Dorschner, M. O., Hawrylycz, M., Humbert, R., Wallace, J. C., Shafer, A., Kawamoto, J., Mack, J., Hall, R., Goldy, J., Sabo, P. J., Kohli, A., Li, Q., McArthur, M., and Stamatoyannopoulos, J. A. (2004) High-throughput localization of functional elements by quantitative chromatin profiling. Nature methods 1, 219–225.
Crawford, G. E., Davis, S., Scacheri, P. C., Renaud, G., Halawi, M. J., Erdos, M. R., Green, R., Meltzer, P. S., Wolfsberg, T. G., and Collins, F. S. (2006) DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays. Nature methods 3, 503–509.
Sabo, P. J., Kuehn, M. S., Thurman, R., Johnson, B. E., Johnson, E. M., Cao, H., Yu, M., Rosenzweig, E., Goldy, J., Haydock, A., Weaver, M., Shafer, A., Lee, K., Neri, F., Humbert, R., Singer, M. A., Richmond, T. A., Dorschner, M. O., McArthur, M., Hawrylycz, M., Green, R. D., Navas, P. A., Noble, W. S., and Stamatoyannopoulos, J. A. (2006) Genome-scale mapping of DNase I sensitivity in vivo using tiling DNA microarrays. Nature methods 3, 511–518.
Crawford, G. E., Holt, I. E., Whittle, J., Webb, B. D., Tai, D., Davis, S., Margulies, E. H., Chen, Y., Bernat, J. A., Ginsburg, D., Zhou, D., Luo, S., Vasicek, T. J., Daly, M. J., Wolfsberg, T. G., and Collins, F. S. (2006) Genome-wide mapping of DNase hypersensitive sites using massively parallel signature sequencing (MPSS). Genome research 16, 123–131.
Ohnesorg, T., Eggers, S., Leonhard, W. N., Sinclair, A. H., and White, S. J. (2009) Rapid high-throughput analysis of DNaseI hypersensitive sites using a modified MLPA approach. BMC Genomics 10, 412.
Schouten, J. P., McElgunn, C. J., Waaijer, R., Zwijnenburg, D., Diepvens, F., and Pals, G. (2002) Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic acids research 30, e57.
Lalic, T., Vossen, R. H., Coffa, J., Schouten, J. P., Guc-Scekic, M., Radivojevic, D., Djurisic, M., Breuning, M. H., White, S. J., and den Dunnen, J. T. (2005) Deletion and duplication screening in the DMD gene using MLPA. Eur J Hum Genet 13, 1231–1234.
Zeng, F., Ren, Z. R., Huang, S. Z., Kalf, M., Mommersteeg, M., Smit, M., White, S., Jin, C. L., Xu, M., Zhou, D. W., Yan, J. B., Chen, M. J., van Beuningen, R., Huang, S. Z., den Dunnen, J., Zeng, Y. T., and Wu, Y. (2008) Array-MLPA: comprehensive detection of deletions and duplications and its application to DMD patients. Human mutation 29, 190–197.
Aten, E., White, S. J., Kalf, M. E., Vossen, R. H., Thygesen, H. H., Ruivenkamp, C. A., Kriek, M., Breuning, M. H., and den Dunnen, J. T. (2008) Methods to detect CNVs in the human genome. Cytogenetic and genome research 123, 313–321.
Bayley, J. P., Grimbergen, A. E., van Bunderen, P. A., van der Wielen, M., Kunst, H. P., Lenders, J. W., Jansen, J. C., Dullaart, R. P., Devilee, P., Corssmit, E. P., Vriends, A. H., Losekoot, M., and Weiss, M. M. (2009) The first Dutch SDHB founder deletion in paraganglioma-pheochromocytoma patients. BMC Med Genet 10, 34.
Birney, E., Stamatoyannopoulos, J. A., Dutta, A., Guigo, R., Gingeras, T. R., Margulies, E. H., Weng, Z., Snyder, M., Dermitzakis, E. T., Thurman, R. E., Kuehn, M. S., Taylor, C. M., Neph, S., Koch, C. M., Asthana, S., Malhotra, A., Adzhubei, I., Greenbaum, J. A., Andrews, R. M., Flicek, P., Boyle, P. J., Cao, H., Carter, N. P., Clelland, G. K., Davis, S., Day, N., Dhami, P., Dillon, S. C., Dorschner, M. O., Fiegler, H., Giresi, P. G., Goldy, J., Hawrylycz, M., Haydock, A., Humbert, R., James, K. D., Johnson, B. E., Johnson, E. M., Frum, T. T., Rosenzweig, E. R., Karnani, N., Lee, K., Lefebvre, G. C., Navas, P. A., Neri, F., Parker, S. C., Sabo, P. J., Sandstrom, R., Shafer, A., Vetrie, D., Weaver, M., Wilcox, S., Yu, M., Collins, F. S., Dekker, J., Lieb, J. D., Tullius, T. D., Crawford, G. E., Sunyaev, S., Noble, W. S., Dunham, I., Denoeud, F., Reymond, A., Kapranov, P., Rozowsky, J., Zheng, D., Castelo, R., Frankish, A., Harrow, J., Ghosh, S., Sandelin, A., Hofacker, I. L., Baertsch, R., Keefe, D., Dike, S., Cheng, J., Hirsch, H. A., Sekinger, E. A., Lagarde, J., Abril, J. F., Shahab, A., Flamm, C., Fried, C., Hackermuller, J., Hertel, J., Lindemeyer, M., Missal, K., Tanzer, A., Washietl, S., Korbel, J., Emanuelsson, O., Pedersen, J. S., Holroyd, N., Taylor, R., Swarbreck, D., Matthews, N., Dickson, M. C., Thomas, D. J., Weirauch, M. T., Gilbert, J., Drenkow, J., Bell, I., Zhao, X., Srinivasan, K. G., Sung, W. K., Ooi, H. S., Chiu, K. P., Foissac, S., Alioto, T., Brent, M., Pachter, L., Tress, M. L., Valencia, A., Choo, S. W., Choo, C. Y., Ucla, C., Manzano, C., Wyss, C., Cheung, E., Clark, T. G., Brown, J. B., Ganesh, M., Patel, S., Tammana, H., Chrast, J., Henrichsen, C. N., Kai, C., Kawai, J., Nagalakshmi, U., Wu, J., Lian, Z., Lian, J., Newburger, P., Zhang, X., Bickel, P., Mattick, J. S., Carninci, P., Hayashizaki, Y., Weissman, S., Hubbard, T., Myers, R. M., Rogers, J., Stadler, P. F., Lowe, T. M., Wei, C. L., Ruan, Y., Struhl, K., Gerstein, M., Antonarakis, S. E., Fu, Y., Green, E. D., Karaoz, U., Siepel, A., Taylor, J., Liefer, L. A., Wetterstrand, K. A., Good, P. J., Feingold, E. A., Guyer, M. S., Cooper, G. M., Asimenos, G., Dewey, C. N., Hou, M., Nikolaev, S., Montoya-Burgos, J. I., Loytynoja, A., Whelan, S., Pardi, F., Massingham, T., Huang, H., Zhang, N. R., Holmes, I., Mullikin, J. C., Ureta-Vidal, A., Paten, B., Seringhaus, M., Church, D., Rosenbloom, K., Kent, W. J., Stone, E. A., Batzoglou, S., Goldman, N., Hardison, R. C., Haussler, D., Miller, W., Sidow, A., Trinklein, N. D., Zhang, Z. D., Barrera, L., Stuart, R., King, D. C., Ameur, A., Enroth, S., Bieda, M. C., Kim, J., Bhinge, A. A., Jiang, N., Liu, J., Yao, F., Vega, V. B., Lee, C. W., Ng, P., Shahab, A., Yang, A., Moqtaderi, Z., Zhu, Z., Xu, X., Squazzo, S., Oberley, M. J., Inman, D., Singer, M. A., Richmond, T. A., Munn, K. J., Rada-Iglesias, A., Wallerman, O., Komorowski, J., Fowler, J. C., Couttet, P., Bruce, A. W., Dovey, O. M., Ellis, P. D., Langford, C. F., Nix, D. A., Euskirchen, G., Hartman, S., Urban, A. E., Kraus, P., Van Calcar, S., Heintzman, N., Kim, T. H., Wang, K., Qu, C., Hon, G., Luna, R., Glass, C. K., Rosenfeld, M. G., Aldred, S. F., Cooper, S. J., Halees, A., Lin, J. M., Shulha, H. P., Zhang, X., Xu, M., Haidar, J. N., Yu, Y., Ruan, Y., Iyer, V. R., Green, R. D., Wadelius, C., Farnham, P. J., Ren, B., Harte, R. A., Hinrichs, A. S., Trumbower, H., Clawson, H., Hillman-Jackson, J., Zweig, A. S., Smith, K., Thakkapallayil, A., Barber, G., Kuhn, R. M., Karolchik, D., Armengol, L., Bird, C. P., de Bakker, P. I., Kern, A. D., Lopez-Bigas, N., Martin, J. D., Stranger, B. E., Woodroffe, A., Davydov, E., Dimas, A., Eyras, E., Hallgrimsdottir, I. B., Huppert, J., Zody, M. C., Abecasis, G. R., Estivill, X., Bouffard, G. G., Guan, X., Hansen, N. F., Idol, J. R., Maduro, V. V., Maskeri, B., McDowell, J. C., Park, M., Thomas, P. J., Young, A. C., Blakesley, R. W., Muzny, D. M., Sodergren, E., Wheeler, D. A., Worley, K. C., Jiang, H., Weinstock, G. M., Gibbs, R. A., Graves, T., Fulton, R., Mardis, E. R., Wilson, R. K., Clamp, M., Cuff, J., Gnerre, S., Jaffe, D. B., Chang, J. L., Lindblad-Toh, K., Lander, E. S., Koriabine, M., Nefedov, M., Osoegawa, K., Yoshinaga, Y., Zhu, B., and de Jong, P. J. (2007) Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature 447, 799–816.
Acknowledgments
We would like to thank Professor Andrew Sinclair for helpful discussions. SE is supported by a University of Melbourne Postgraduate Research Scholarship. S.J.W. is funded by NHMRC Australia (grant numbers 491293 and 546478).
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Ohnesorg, T., Eggers, S., White, S.J. (2012). Detecting DNaseI-Hypersensitivity Sites with MLPA. In: Deplancke, B., Gheldof, N. (eds) Gene Regulatory Networks. Methods in Molecular Biology, vol 786. Humana Press. https://doi.org/10.1007/978-1-61779-292-2_12
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DOI: https://doi.org/10.1007/978-1-61779-292-2_12
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