Question

Write no more than necessary to answer the question. Pictures are encouraged if they would be helpful. For all questions, you may assume that high quality reagents are available and all relevant DNA sequences have been cloned. Money is no object, but experiments should be focused on answering the question. 

You are a postdoc at the Jedi Institute for Advanced Studies. Your project is focused on the cellular pathway regulating sensitivity to the Force. Pedigree analysis suggests that Force sensitivity runs in families and is inherited in a Mendelian dominant fashion. Classical genetic and mapping studies performed during the Old Republic localized the gene for Force sensitivity to the very end of the P arm of chromosome 3 (locus C3p0).  Using bioinformatics analysis, a collaborator has discovered a polymorphism in one of the open reading frames in this region (provisionally named FORCE) that correlates strongly with sensitivity to the Force. 

2. You hypothesize that the wild type FORCE protein represses genes that are involved in the signaling pathway that responds to the Force. The mutation results in the abnormal upregulation of these genes, and thus increased sensitivity to stimulation of the pathway. 

D. Describe an experiment to determine which regions of the genome are bound by the wild type FORCE protein

Answer 1

2D. To determine which regions of the genome are bound by the FORCE protein, we can perform many experiments
1. Perform SELEX to identify the binding sequence of the FORCE protein. Perform a bioinformatics search to identify the location of these binding sites in the genome.
Perform a microarray assay between the WT and the mutant cells.
Search of the FORCE binding motif in the differentially regulated genes.
We can confirm whether FORCE indeed binds to these regions by EMSA and ChIP experiments

2. We can perform ChiP-Seq analysis using the Anti-FORCE antibody.
It will provide all the sites bound by the FORCE protein in the cell.
In this method, the FORCE protein is allowed to bind to its cognate binding site and cross-linked to stabilize the interaction.
The DNA is sheared and subjected to DNase cleavage. The FORCE-bound regions are protected from the cleavage and can be amplified in the PCR.