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 ampli fied 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 proteinbinding sites in a DNA fragment. The method is applicable to the identi fication of base changes, e.g., single-nucleotide polymorphisms (SNPs), that eliminate protein binding to DNA. © Springer Science+Business Media New York 2013.