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DNA affinity chromatography

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Abstract

DNA-affinity chromatography has been used for the purification of DNA-binding proteins that control various cellular processes. There have been improvements in coupling methods and choice of supports over the years. The procedure for coupling 5′-aminoethyl-(dT)18 to silica activated with N-hydroxysuccinimide and a carbodiimide has been described. Also, the cyanogen bromide mediated coupling of aminoethyl-(dT)18 to Sepharose is described. Determination of (dT)18-coupling to silica and Sepharose is by 5′ end-labeling an oligonucleotide containing a (dA)18 stretch of sequence and determining how much hybridizes with the (dT)18 support. Enzymatic synthesis of a double-stranded DNA-silica or Sepharose prevents modification of nucleotide bases. We have explained the use of DNA and RNA templates for template-directed enzymatic synthesis of affinity columns. DNA-affinity chromatography is a powerful method with broad applicability and we are currently extending this technology for purifying transcription factors, polymerases, and nucleases.

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Authors and Affiliations

  1. Department of Biochemistry, University of Tennessee, 858 Madison Avenue, 38163, Memphis, TN

    Harry W. Jarrett

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  1. Priya Sethu Chockalingam
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  2. Luis A. Jurado
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  3. Harry W. Jarrett
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Correspondence to Harry W. Jarrett.

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Chockalingam, P.S., Jurado, L.A. & Jarrett, H.W. DNA affinity chromatography. Mol Biotechnol 19, 189–199 (2001). https://doi.org/10.1385/MB:19:2:189

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