Two traditional methods, DNA protection assay (DNA Footprint) 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 and a polyacrylamide gel, the DNA is labeled with a fluorescent dye and the resulting fragments from the methods above are analyzed with the 3730 DNA Analyzer automated capillary electrophoresis instrument at the Facility. In addition, non-radioactive gel shift assays are also commonly performed along with DNA Footprinting, which the Facility can also provide.
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.
References and Resources
- Example of MSACE results
- Procedures and recommendations for MSACE [pdf]
- merighi-etal_2006_JBac_(188)5089-5100 [pdf]
Molecular Characterization of Pantoea stewartii HrpY, a Conserved Response Regulator of the Hrp Type III Secretion System, and its Interaction with the hrpS Promoter
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 at a promoter for a gene. The protein of interest provides protection to specific nucleotides resulting in fewer DNA fragments in this area after exposure to a DNA degradation procedure, e.g. DNaseI exonuclease. The resulting fragment patterns are 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.
References and Resources
- Example of DFACE results
- Procedures and recommendations for DFACE [pdf]
- joshi-et-al_2012_JBact_(194)1350-1360 [pdf]
- wang-etal_2007_JBac_(189)4880-4886 [pdf]
- zianni-et-al_2006_JBT_(17)103-113 [pdf]
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 the binding experiment by utilizing stains specific for protein (SYPRO Ruby) and DNA (SYBR Green) (Reference) in the same gel. Alternatively the DNA can be imaged with the fluorescent tag instead of SYBR green before the protein stain. The gels are imaged with a Versadoc 4000 MP from BioRad.
How to order services:
- Log into dnaLIMS (or create an account if you have not done so previously),
- Click on "Other Services" tab
- Click on the "Promoter Characterization" Order Form link
- Follow the instructions.
How to deliver samples
Instructions to deliver samples from both campus and non-campus clients.
How to obtain results
- An email will be sent when the results are available on line through dnaLIMS.
- Log in and click on "Download Fragment Analysis Results" to obtain the .fsa (electropherogram) files for DFACE and MSACE results.
- Click on the "downlab" tab, and select the appropriate order # to download the excel and image files which contain the analysis of the electropherograms and gels from EMSA.
The facility has the following software to aid in the analysis of the data generated by the 3730 DNA Analyzer (*.fsa files) by the Facility staff: GeneMapper and GeneMarker. In addition, there are 3 free programs available for analyzing the electropherogram files with varying levels of features:
[pdf] - Some links on this page are to .pdf files. These are designated by [pdf] following the link. If you require these materials in alternate format contact our web manager.