18-07-2012, 01:47 PM
DNA Footprinting with an automated DNA Analyzer
DNAFootprinting.pdf (Size: 2.42 MB / Downloads: 117)
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.
Fluorescent Primer Design
For use in: (1) the DNA sequencing reactions which act as a
standard/ladder and (2) the PCR reaction to make the DNA probe
Used routine DNA sequencing guidelines:
18 -25mer.
Tm at 55 – 60C.
about 50% GC.
Primer purified by HPLC
5’ label with FAM or VIC which worked well; HEX is okay
DNA sequencing reaction and Analysis
DNA probe made by PCR performed with labeled primers followed by a
cleaned up protocol, e.g. Qiagen PCR column
Used ThermoSequenase Dye Primer DNA Sequencing Kit (USB Corp.)
with the manufacturers protocol but at maximal values and amounts.
For each base/tube of a sequencing reaction used 0.2 to 2ul in 10ul
HiDi and 0.1ul LIZ 500 standards (Applied Biosystems)
Analyze with 3730 DNA Analyzer (Applied Biosystems) using default
Genemapper50-POP7-1 Run module, but in addition increased
injection voltage to 3kV and 30sec from 1.5kV and 15sec respectively
to increase signals
Detailed DNA footprint protocol for HrpY
-type activator that has a Helix-turn-helix motif that binds to the hrpS
promoter of the phytopathogen Pantoea steweartii.
Part of a two component system that is necessary for sensing a host, maize,
and activating the expression of the hrp proteins which are necessary for
infection
DNA fragment for digestion
One primer labeled with 6-FAM; 386bp long
PCR protocol: 25x of 30sec/95C – 30sec/50C – 60sec/72C
PCR products were purified from gel and quantified by UV spectrophotometry.