title: Detection of regulatory polymorphisms: high-throughput capillary DNase I footprinting.
abstract: We describe a method for high-throughput analysis of protein-binding sites in DNA using 96-well plates and capillary electrophoresis. The genomic DNA or plasmid DNA to be analyzed is amplified using fluorescent primers, incubated with an appropriate nuclear extract and treated with DNase I. Separation of the DNase I-generated fragments and co-analysis of their base sequences identify the position of protein-binding sites in a DNA fragment. The method is applicable to the identification of base changes, e.g., single-nucleotide polymorphisms (SNPs), that eliminate protein binding to DNA.
authors.affiliation: Institute of Structural and Molecular Biology, University College London, London, UK.
authors.forename: Matthew
authors.initials: M
authors.lastname: Hancock
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Shephard
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002725:Chloroform / Q000737:chemistry; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000379:methods*; D019800:Phenol / Q000737:chemistry; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D023302:Protein Footprinting / Q000379:methods*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D012822:Silicon Dioxide / Q000737:chemistry
chemical_list: D019800:Phenol; D012822:Silicon Dioxide; D002725:Chloroform; D004247:DNA; D003850:Deoxyribonuclease I