Services - Promoter Characterization

Two traditional methods, DNA protection assay and primer extension, that are used to determine promoter binding sites and transcription start sites respectively can be done at the Facility. Instead of using radioactivity the DNA is labeled with a fluorescent dye and the resulting fragments from the methods above can be analyzed with instrumentation at the Facility.

Transcription Start site

Primer Extension analysis typically results in one cDNA product which can be compared to DNA sequencing reaction extension products in order to determine the last base of the cDNA and therefore the first base of the mRNA for a particular gene. The analysis can be performed on the Facility's 3730 DNA Analyzer (Applied Biosystems) which can analyze DNA fragments labeled with a fluorescent dye to determine the size of the fragment with a resolution of one base. With 48 capillaries the instrument can analyze up to 48 samples simultaneously as well as detect 5 different fluorescent dyes therefore allowing samples to be multiplexed in a single capillary.

Protein-DNA binding site

A DNA protection assay, commonly known as a DNase Footprint Assay, is used to determine the binding site of a protein regulator to DNA which is typically a promoter for a gene. The protein of interest provides protection to specific nucleotides resulting in fewer DNA fragments in this area after the digestion step. The resulting fragment pattern can be analyzed at the Facility with the 3730 DNA Analyzer and compared to DNA sequencing reaction products in order to determine the exact bases which interact with the protein.

Typically before a DNA protection Assay is done Electrophoretic Mobility Shift Assays (EMSA) are performed in order to demonstrate that the protein - DNA binding is specific and competitive. The Facility can help with this as well by imaging the gel after utilizing stains specific for protein (SYPRO Ruby) and DNA (SYBR Green) (Reference). The gels are imaged with a Versadoc 4000 MP from BioRad.

The facility has the following software to aid in the analysis of the data generated by the 3730 DNA Analyzer (*.fsa files): GeneMapper 4.0.

Peak Scanner ver 1.0 is a free program that can be used to view, edit and print data from *.fsa files which is the Applied Biosystems' proprietary file format.


Procedures and Recommendations:
Transcription Start Site [pdf file]
Protein-DNA binding site [pdf file]
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