I need to summarize this section of a research paper on
materials and mthods. I have no idea wat it is even saying. please
explain and summarize the idea of the method if you can.:
DNA curvature prediction and electrophoretic mobility shift
assays.TheupaCpromoter region was analyzedin silicousing bend.it,
aprogram that enables the prediction of a curvature propensity plot
calcu-lated with DNase I-based parameters
(http://hydra.icgeb.trieste.it/dna/)(73). The curvature is
calculated as a vector sum of dinucleotide geome-tries (roll, tilt,
and twist angles) and expressed as degrees per helical
turn(10.5°/helical turn1°/bp). Experimentally tested curved motifs
producecurvature values of 5 to 25°/helical turn, whereas straight
motifs give val-ues below 5°/helical turn. TheupaC250-bp promoter
region was ampli-fied using primers upaC.pro.ext-5+250 and
upaC.pro.ext-3-1ATG, and its intrinsic curvature was assessed by
comparing its electrophoretic mo-bility with that of an unbent
marker fragment (Promega; 100-bp DNAladder) on a 0.5%
Tris-borate-EDTA (TBE), 7.5% PAGE gel at 4°C forretarded gel
electrophoretic mobility.Gel shift assays were performed
essentially as previously described (3).A DNA mixture comprising an
equimolar ratio of the PCR-amplified upaC promoter region and
TaqI-SspI-digested pBR322 was incubated at room temperature for 15
min with increasing amounts of native purified H-NS protein (a gift
from S. Rimsky) in 30l of reaction mixture containing 40 mM HEPES
(pH 8), 60 mM potassium glutamate, 8 mM mag-nesium aspartate, 5 mM
dithiothreitol, 10% glycerol, 0.1% octylphenoxy-polyethoxyethanol,
0.1 mg/ml BSA (H-NS binding buffer). DNAfragments and DNA-protein
complexes were resolved by gel electropho-resis (0.5% TBE, 3% MS
agarose gel run at 50 V at 4°C) and visualized after staining with
ethidium bromide.





