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DNA Footprinting with an Applied Biosystems 3730 DNA Analyzer: does the shoe fit .yes!
6FAM
BSA
6FAM
6FAM
6FAM
6FAM
6FAM
6FAM
6FAM
Capillary Electrophoresis
Development of DNA Foot print Analysis Techniques and Goals of This Study
First performed in 1977, and utilized radioactively labeled DNA, slab gels, and autoradiography In 1994, dyes and fluorescent gel imager were used instead of radioisotopes and autoradiography but the slab gel remained. In 2000, the slab gel and imager were replaced by an automated capillary electrophoresis instrument: 310 DNA Analyzer. In 2004, ...........
Demonstrate the feasibility by reproducing a protection assay previously done with autoradiography and a slab gel Demonstrate that each peak could be accurately identified Apply this method to a previously uncharacterized protein
Primer purified by HPLC 5 label with FAM or VIC which worked well; HEX is okay
A T G C
-58 C C G A T A T C A G C A T A T G A C G A T G G T T -34
BigDye v3.1
gc (-70,-69)
tc (-39,-38)
cc (-11,-10)
a c g (-12,-14,-16)
ag (-42,-43)
gg (-53,-54)
gg (-71,-72)
0.0005 (good)
HrpY DNA footprint analysis with varying concentrations of transcription activator HrpY
Region of Interest
140M HrpY 0g BSA 70M HrpY 6g BSA 35M HrpY 9g BSA 17.5M HrpY 10g BSA 4.3M HrpY 12g BSA 2M HrpY 12g BSA 0M HrpY 12g BSA
II
II
BSA
BSA
Green box protected regions Red box hypersensitive regions I and II are distinct regions that fit binding motif: 1) T- N11A 2) centered about -70 from transcription start site
-126 T A T G T A A C C G T T T T C A T T T C A C A C A G G G T T A G T C A G G A A A T C C T T A C A A T C C T C C A T C -69
Upper panel : red without HrpY; blue - with HrpY Lower panel: red t; green a; blue c; black - g
-68 A A T T C C T T A C C C G A T A T C A G C A T A T G A C G A T G G T T T A T C T G T G C T G A C A T -19
Upper panel : red without HrpY; blue - with HrpY Lower panel: red t; green a; blue c; black - g
DNA primer extension analysis to determine the transcription start site for hrpS gene of Pantoea stewartii
tatgtttacGaagtgcttacagggg
agataaaCcatcg
Two transcription start sites were identified (purple peaks; capitalized text). Red t; green a; black g; blue c.