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Isolation and Sequencing of Short Cell-Surface-Bound DNA

Authors

  • Vera S. Mal'shakova,

    1. Institute of Chemical Biology and Fundamental Medicine, Siberian Division of the Russian Academy of Sciences, Novosibirsk, Russia
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  • Dmitry V. Pyshnyi,

    1. Institute of Chemical Biology and Fundamental Medicine, Siberian Division of the Russian Academy of Sciences, Novosibirsk, Russia
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  • Alexander A. Bondar,

    1. Institute of Chemical Biology and Fundamental Medicine, Siberian Division of the Russian Academy of Sciences, Novosibirsk, Russia
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  • Valentin V. Vlassov,

    1. Institute of Chemical Biology and Fundamental Medicine, Siberian Division of the Russian Academy of Sciences, Novosibirsk, Russia
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  • Pavel P. Laktionov

    1. Institute of Chemical Biology and Fundamental Medicine, Siberian Division of the Russian Academy of Sciences, Novosibirsk, Russia
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Address for correspondence: Vera S. Mal'shakova, Institute of  Chemical Biology and Fundamental Medicine, Siberian Division of the Russian Academy of Sciences, Lavrentiev Ave., 8, Novosibirsk 630090, Russia. Voice: +7-383-3304654; fax: +7-383-3333677. vera_mal@niboch.nsc.ru

Abstract

To isolate the DNA fragments bound to cell-surface proteins with high affinity, peptide–DNA complexes were obtained by mild treatment of human endothelial cells with trypsin. After binding of nucleoprotein complexes to BrCN-Sepharose and extensive washing, 7–11 nt DNA fragments were eluted with acidic buffer, labeled with 32P, and isolated by PAGE. The fragments were ligated to double-stranded oligonucleotide adapters, amplified by PCR, and cloned in pBluescript II KS (+) vector. Sequences of 12 clones containing the insert of cell-surface-bound 7–11-mer DNA fragments were obtained.

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