Question

I am currently studying experimental analytical assays such as:

1. deletion analysis + reporter assay
2. linker scanning mutation assay
3. DNase I foot-printing assay
4. Electrophoretic mobility shift assay
5. ChIp-Seq

And I would like to look further into the applications of these methods. I am particularly comfortable with (1), (3), and (4) so any suggestions on an experiment I could test would be much appreciated. Not looking to do anything too complex but just something interesting and simple. I know that this isn't the typical homework question, but we do have to write about a paper about such an experiment that uses one of these methods.

Answer 1

1. The reporter genes assay is used for analyzing the function of cis-acting genetic elements such as promoters (“promoter bashing”). example deletion analysis of the human factor VIII gene promoter region.
2. 'Linker scanning' is use for systematically surveying a segment of DNA for functional elements by mutagenesis. It can also be used to make site specific mutations at any position of any sequence rapidly and efficiently.
3. DNA footprinting is used to study DNA-protein interaction.
4. An electrophoretic mobility shift assay(EMSA, also known as a gel shift assay) is used to determine if a protein is able to directly interact with a short, specific sequence of DNA. It can be used to study the binding of nuclear proteins to DNA and to characterize how these proteins interact with and modify chromatin to regulate gene expression and, more globally, cell differentiation.
5. ChIP-sequencing. ChIP-sequencing, also known as ChIP-seq, is a method used to analyze protein interactions with DNA. ChIP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. ChIP-seq have been used to map genomic regions and histone modifications.