IngentaConnect DNA affinity chromatography

Authors: Chockalingam, Priya; Jurado, Luis; Jarrett, Harry¹

Source: Molecular Biotechnology, Volume 19, Number 2, October 2001 , pp. 189-199(11)

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.

Keywords: DNA; affinity; chromatography; silica; Sepharose; enzymatic synthesis

Document Type: Research article

DOI: 10.1385/MB:19:2:189

Affiliations: 1: Department of Biochemistry, University of Tennessee, 858 Madison Avenue, 38163, Memphis, TN, Email: hjarrett@utmem1.utmem.edu

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