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. 2012 Apr 10;51(15):3578-81.
doi: 10.1002/anie.201107714. Epub 2012 Feb 16.

Enzymatically incorporated genomic tags for optical mapping of DNA-binding proteins

Soohong Kim  1 , Anna Gottfried, Ron R Lin, Thomas Dertinger, Andrew S Kim, Sangyoon Chung, Ryan A Colyer, Elmar Weinhold, Shimon Weiss, Yuval Ebenstein
Affiliations

Enzymatically incorporated genomic tags for optical mapping of DNA-binding proteins

Soohong Kim et al. Angew Chem Int Ed Engl. .
No abstract available

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Figures

Figure 1
Figure 1
a) Schematic representation of QD-labeled RNAP bound to sequence-specific-labeled T7 bacteriophage DNA. The DNA methyltransferase M.BseCl recognizes specific rare sequences in the T7 genome (5′-ATCGAT-3′) and conjugates an aziridine cofactor 6BAz with an attached biotin (yellow triangle). This biotin group can be used for site-specific labeling with QDs. b) RNAP-bound T7 bacteriophage genome stretched over a polylysine surface. c) Image of flow-stretched, YOYO-1 stained T7 bacteriophage DNA (white) with QD-labeled RNAP (green) and M.BseCl refTags labeled with spectrally distinct QDs (red). Overlapping red and green signals are shown in yellow. d) Conceptual representation of a genome-wide map of promoter and DNA methyltransferase sites (bottom). Histogram of M.BseCl positions represented in red and RNAP positions represented in green (top).
Figure 2
Figure 2
a) Histogram obtained from localizing refTag: QDs on stretched T7 bacteriophage genomes. Red vertical lines represent expected position of recognition sequences. b) Selected images of extended YOYO-1 stained T7 genomes (white) labeled with QDs on M.BseCl DNA MTase refTag sites (red). Scale bar: 3 µm.
Figure 3
Figure 3
Sequence-specific methyltransferase-induced labeling of DNA (SMILing DNA) creates a distinct barcode which reports the preferential binding sites of RNAP on T7 bacteriophage DNA. Selected images of QD-labeled RNAP (green) bound to promoters on stretched YOYO-1 stained T7 bacteriophage DNA (white or blue) having refTag sites labeled with spectrally distinct QDs (red). Overlapping red and green signals are shown in yellow. Scale bar: 3 µm.
Figure 4
Figure 4
Histograms for localized RNAP on T7 bacteriophage using distance measurment to the DNA ends (left) versus localization by refTags (right). a), b) Histograms of all analyzed strands with Gaussian fit. c), d) Histograms based on selection of long strands (>80 pixels) with Gaussian fit. Dotted lines represent actual position of Φ13 promoter. Histograms using refTags yield a fivefold increase in accuracy as evidenced by sharp reductions in the width of promoter localization distributions.
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References

    1. Shendure J, Ji H. Nat. Biotechnol. 2008;26:1135–1145. - PubMed
    1. Neely RK, Deen J, Hofkens J. Biopolymers. 2011;95:298–311. - PubMed
    1. Meng X, Benson K, Chada K, Huff EJ, Schwartz DC. Nat. Genet. 1995;9:432–438. - PubMed
    1. Michalet X. Science. 1997;277:1518–1523. - PubMed
    1. Chan EY, Goncalves NM, Haeusler RA, Hatch AJ, Larson JW, Maletta AM, Yantz GR, Carstea ED, Fuchs M, Wong GG, et al. Genome Res. 2004;14:1137–1146. - PMC - PubMed

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