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Quantitative Analysis of Protein-DNA Associations in Vivo Using Real-Time PCR, Rev A

Accelrys), and are presented in the table. To demonstrate that promoter occupancy was independent of the specific region of the promoter amplified, we examined two different regions of the glucagon promoter, designated as Gluc(1) and Gluc(2). These two regions are located approximately 300 bp apart in the proximal glucagon promoter. For real-time PCR reactions, 3 l of each immunoprecipitated sample was used as template in 25 l reactions containing 3 mM MgCl2, 0.25 mM dNTPs, 10 pmol primers, 0.5 U Platinum Taq DNA polymerase (Invitrogen), and a 1:75,000 dilution of SYBR Green I dye (Molecular Probes). Thermal cycling was performed for 40 cycles using an iCycler iQ system, and continuous SYBR Green I fluorescence monitoring was performed according to Bio-Rad protocols. Cycling parameters for all amplifications were 30 sec at 95C, 30 sec at 55C, and 30 sec at 72C, for 30 cycles. Amplified promoter fragments were subcloned into the T/A cloning vector pCR2.1 (Invitrogen) according to the manufacturers protocol, and 34 clones from each PCR reaction were sequenced to confirm the identity of the amplified fragment.

The relative proportions of coimmunoprecipitated promoter fragments were determined based on the threshold cycle (CT) value for each PCR reaction. The CT value is defined as the cycle at which fluorescence rises to 10 times above the mean standard deviation of the background levels in all reaction wells. For every promoter studied, a ΔCT value was calculated for each sample by subtracting the CT value for the input (sample prior to immunoprecipitation) from the CT value obtained for the immunoprecipitated sample. A Δ
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