DNA affinity chromatography.
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.
Chockalingam
P S
PS
Department of Biochemistry, University of Tennessee, 858 Madison Avenue, Memphis, TN 38163, USA.
Jurado
L A
LA
Jarrett
H W
HW
D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D004247:DNA / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D012685:Sepharose / Q000737:chemistry; D013941:Thymine
D004268:DNA-Binding Proteins; D012313:RNA; D004247:DNA; D012685:Sepharose; D013941:Thymine