Mapping Instrumentation 



DNA-Protein-Binding Assay — Use of gel retardation for recognition of 

 promoter sequences that are necessary for the polymerase enzyme to 

 synthesize DNA for sequencing studies. Increasing quantities of T7 RNA 

 polymerase were incubated withi pET-1 DNA (contains a strong T7 promoter) and 

 pAT153 DNA (no promoter) for 10 min at room temperature; samples were then 

 electrophioresed in a 1% agarose gel for 2 hr at 2.5 V/cm. As the ratio of 

 polymerase molecules to DNA increased, the quantity of pET-1 DNA band 

 decreased, and a new band with lower mobility appeared. Without requiring 

 quantitative loading of the gel. the ratio of fluorescence in the pET-1 band to that 

 in the control pAT153 band permits quantitation of the fraction of the pET-1 

 molecules bound to polymerase. This is a useful technique for finding specific 

 promoter sequences; once found, these promoter sequences can be attached to 

 DNA fragments of choice (e.g.. fragments the researcher is interested in 

 sequencing). (Photograph provided by Betsy Sutherland, Brookhaven National 

 Laboratory.) 



