Procedures and Recommendations for DFACE:
DNA Footprint Analysis by Automated Capillary Electrophoresis
Plant-Microbe Genomics Facility
Ohio State University
Biological Sciences Building, Room 420
484 W. 12th Ave., Columbus OH 43210-1214
Telephone: 614/247-6204
FAX: 614/247-8696
E-mail: [email protected]
Website: www.pmgf.osu.edu
Version 1.1
February, 2016
1. Introduction
1.1. Overview: A DNA protection assay, commonly known as a DNA Footprint assay,
is done by digesting the DNA in the presence of a protein in order to show the
specific bases in which the protein interacts with the DNA. This is commonly
done to interrogate promoters and the proteins that regulate gene expression.
Originally the bases that contact the protein(s) and their relative concentrations
were determined by gel electrophoresis and autoradiography with protected
regions displaying bands of lower intensity or no bands at all. In addition, a DNA
sequencing reaction was run in parallel on the same gel in order to determine the
3’ base of each fragment.
The Facility can do the same experiment by replacing the radioactive isotope and
gel electrophoresis/autoradiography with a fluorescent dye and automated
capillary electrophoresis, respectively. In summary, the client provides the dyelabeled DNA digestion product along with template and primer for the DNA
sequencing reaction, and the Facility will then analyze the DNA fragment pattern
with a 3730 DNA Analyzer to determine the protein contact areas as well as
identify the base. Following is the reference for an example of this type of
analysis performed at the Facility: Zianni, et al (2006) Journal of Biomolecular
Techniques. 17: 103-113. See Figure 1 for a schematic explanation of DFACE.
1.2. Pre-experimental Considerations: Prior to the Facility starting any work, we
request two things: (1) a free face-to-face consultation or, at the minimum, a
direct phone consultation and (2) a completed online order at dnaLIMS for each
request of services. This initial communication is necessary to clarify the
customer’s goals. In order for the Facility to best serve the client, it is essential
that the client have a clear understanding of the service’s capabilities and the
input required. A critical point that must be made clear is that optimization is
almost inevitable due to the specific characteristics of diverse samples and the
requirements of individual customers. There are, however, steps that can be
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taken during sample preparation to minimize the need for lengthy optimization.
The following are guidelines and recommendations, as well as specific
information, provided to maximize the value of the Facility’s services. The
guidelines have been provided for the benefit of the customer; they are, of
course, not strict rules.
1.3. Diagram of DFACE:
Figure 1. Schematic diagram of DFACE. DNA probe labeled with a fluorescent
dye (6FAM) is incubated with the hypothesized DNA binding protein(s) or a
control protein such as bovine serum albumin (BSA) to show specificity. The
DNA is digested with an endonuclease resulting in a population of DNA
fragments which are separated, and ordered based upon size by automated
capillary electrophoresis. By comparing the two resulting fragment patterns a
region in which the peaks are missing or reduced in amount indicates a
protection site and therefore a putative interaction between the DNA and protein
of interest.
2. DNA Foot print Analysis and DNA Sequencing
2.1. Primer and Probe Design: The probe (DNA) has to be labeled with a covalently
attached fluorescent dye, and the easiest way to do this is by ordering the
oligonucleotide that is used to generate the amplicon (probe) with the appropriate
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dye attached. The best dyes to use are FAM and/or VIC, and ordering
oligonucleotides from Applied Biosystems is recommended to insure quality and
compatibility with the DNA Analyzer. Other dyes and manufacturers can be used
but have disadvantages, so please contact the Facility if you would like to use
different dyes. Both of the oligonucleotides can be labeled with one of each of the
dyes above which would allow simultaneous interrogation of both strands of the
probe, although labeling one strand of DNA can work as well. Since the sample
processing and electrophoresis can be multiplexed, thereby accommodating both
strands simultaneously, the only additional cost to doing both strands would be
the doubling of analysis time. The first 40 bases of the probe (amplicon) do not
resolve particularly well on the DNA analyzer, and can be hard to interpret for
protection sites. By labeling both strands there is the added benefit of generating
fragments from the entire probe in the size range that are well resolved during
electrophoresis.
The DNA probe can be from 100 to 600 bases long, although when 400 to 600
bases in length this can be more challenging due to the sequencing and
digestion requirements. It might be possible to attempt a longer probe up to 1200
bases, but is has not been verified.
2.2. Digestion Protocol: It is not possible to recommend a specific protocol since
there are so many different proteins that have quite varied optimum binding
conditions. None the less, it is possible to provide some general guidance.
Demonstrating binding and specificity is a critical step prior to DFACE and is
usually done by employing an electrophoretic mobility shift assay (EMSA). A
good starting procedure for EMSA can be found here: http://pmgf.osu.edu/.
A good starting point for the digestion optimization is utilizing the same conditions
as were used for protein/DNA incubation step during EMSA, followed by
digestion with DNase I according to the manufacturer’s suggestion. This step
always requires optimization. The optimal digestion protocol can be adjusted with
time of incubation or DNase I concentration, but most clients have had better
results by changing the latter. So we recommend a broad range of DNase I
concentrations, such as 1000-fold, to determine the optimal concentration
initially, and then narrowing it from there based upon the results. The best way to
stop the DNase I digestion is incubation at 78°C for 10 min or whatever heat
based protocol the endonuclease manufacturer recommends.
Establishing specificity is critical, and this is often times done by employing a
non-specific probe, such as dIdC, and/or non-specific protein, such as Bovine
serum albumin (BSA). Use of BSA as a negative control is recommended since
the presence of any protein in the digestion reaction can affect the digestion
pattern. In cases where negative controls with no proteins were compared to
experimental samples, the fragment patterns were so different as to be noncomparable. When the same experiment was done with the probe incubated with
BSA vs. the DNA binding protein the DNA fragment patterns were very
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comparable. It is suggested to use BSA as a non-specific control, such that it is
at the same concentration as the protein of interest and in the same buffer to
minimize the possible differences in digestion conditions.
As for probe concentration, clients have used 1 – 1000 ng in the reaction,
although most have used in the range of 10 – 100 ng per digestion. The buffer
content and protein concentration are very different for every different protein, so
useful recommendations cannot be made other than to look to the literature for
information.
The optimization protocol can be checked on an agarose gel optimized for small
fragment visualization, e.g. 2 to 2.4%. The DNA can be imaged by the use of the
attached fluorescent dye as many imagers have LED/Laser light sources and
filters specific to FAM and VIC. Figure 2 is an example of such an optimization
that shows an overdigested pattern in lane 1, as made evident by the presence of
only digested fragments (D) and under digested probe in lane 4 in which most of
the probe (I) is still intact or only slightly digested (D). The object is to have both
intact probe as well as digested fragments such as in lane 2 and 3.
Figure 2. Agarose gel with DNA probe
digested at various amounts of DNAase I. 1 is
100 µg, 2 is 10 µg, 3 is 1 µg, and 4 is 0.1 µg of
DNase I. S is a 100bp ladder starting at
100bp. I is intact probe, and D is the digested
DNA fragments.
Figure 3 demonstrates a different probe from that shown in Figure 2, but the
client chose to analyze the digestion optimization products on the 3730 DNA
analyzer instead of an agarose gel although with similar results. Please note that
the intact probe peak is just visible at the right hand edge of electropherograms C
and D, but not A or B. A is over digested as there is no peak for the intact probe
or any detectable peaks. B is over digested as well, with many small peaks being
visible but not larger peaks. In addition the overall height of the peaks trends
downward. C represents an appropriately digested sample in which the general
peak heights are the same across the length of the probe, there is intact probe
present, and nearly every base is represented by a fragment in the pattern. D is
under digested as there are few fragments but the intact probe is present.
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A
B
C
D
Figure 3. Electropherograms of FAM labeled DNA that was digested with various
amounts of DNase I. A is 50, B is 5, C is 0.5, and D is 0.05 milliKunitz units of
DNase I.
2.3. DNA Fragment Purification: The digested DNA must be purified to remove the
protein and buffers from the digestion reaction, so a solid phase extraction kit
(such as Qiagen MinElute Reaction Cleanup Kit) is recommended. The kit should
be for PCR products, and retain fragments down to 40 bp in size. Elute the DNA
with water in as minimal a volume as possible, since buffer and larger volumes
can lead to reduced signal during electrophoresis.
2.4. Template Design: A template is necessary for the DNA sequencing reaction and
includes all of the sequence from the DNA probe. The template can be either
cloned DNA in a plasmid or a purified PCR product. This type of DNA sequencing
reaction can read about 500 bases, so this size maximizes the analysis
capabilities of the kits and instruments. It should be possible to extend the
reaction beyond 600 bases, but it has not been verified. If you decide to use a
PCR product as the template, then the same primer from above can be used to
generate the PCR product although the primers should not be labeled with a dye.
A DNA sequencing reaction will be performed for each of the primers that are
labeled with a dye utilizing the same template for each reaction.
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2.5. DNA Sequencing Reaction: The DNA sequencing reaction will be performed by
the Facility with the Thermo Sequenase Dye Cycle Sequencing Kit (Affymetrix;
P/N 78500) according to the manufacturer’s protocol.
3. Submission of Samples
3.1. Samples: Send the dye-labeled primer(s) at a concentration of 2 µM (2
pmoles/µL) in a total volume of 10 µL. If providing a plasmid for sequencing, then
send 1 µg at a concentration of 50 to 200 ng/µL. If providing a PCR product for
sequencing, then send 20 ng at a concentration of 2 to 5 ng/µL. The primer(s),
plasmid, and/or PCR product can be shipped in water or 10mM Tris, pH 8.0.
Please provide 10 to 20 µL of each digestion/protection assay in water. Send all
the samples in 0.6 or 1.5 mL microcentrifuge tubes that are clearly labeled with
the name, concentration, date, and your initials. In addition we require the
information described below in order to properly analyze the samples. Please
send printed versions along with the samples and electronic versions by email to
the following address: [email protected].
3.1.1. Primer: sequence, Tm, dye, concentration, volume
3.1.2. Template: plasmid or PCR product, concentration, volume, size (bp),
sequence with map that includes such features such promoter region,
translation start codon, and primer location etc.
3.1.3. List the name of each sample that also describes relative treatment such
as presence of protein, digestion condition, and negative or positive control.
3.1.4. List the combination of samples that are to be compared during the
analysis of the electropherograms.
3.2. Shipment: Tubes should be wrapped in parafilm to prevent the caps from
opening during transit. Samples have been sent successfully by overnight courier
at ambient temperature, but we recommend shipping samples on wet or dry ice.
For all shipment methods the tubes should be in a protective layer, such as
bubble wrap, to prevent physical damage to the tubes from handlers, ice blocks,
etc. A completed order form from dnaLIMS must be included with each order. All
tubes and their samples names must match exactly the names on the order form.
Ship the samples to:
Plant-Microbe Genomics Facility
484 W. 12th Avenue
Room 420
Columbus OH 43210-1214
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3.3. Client Account: For first time clients, please create an account in dnaLIMS. This
is required for you to receive your data, and must be done prior to processing the
samples at the Facility. Please place orders through the online software as
follows:
a. Login to dnaLIMS.
b. Select the “Other Services” tab at the top left of the page.
c. Select “Promoter Characterization” under the heading “Order Forms”
d. Select “DFACE” from the dropdown menu
e. Enter your number of samples and follow the rest of the directions on screen.
f. Contact PMGF if you have any issues.
4. Data and Analysis
4.1. Software: The extension products from the DNA sequencing reaction and the
DNA fragment pattern will be analyzed with the 3730 DNA Analyzer (Applied
Biosystems) followed by data analysis with the software GeneMapper. By
overlaying the appropriate electropherograms with GeneMapper the peaks
patterns can be read manually to determine the DNA sequence and the
fragments that show protection or hypersensitivity. Typically the analysis is done
with the client present in order to facilitate data interpretation, but this is not
necessary.
4.2. Data Format: The files produced by the 3730 DNA Analyzer (*.fsa) will be
available on line through dnaLIMS and have a proprietary format, therefore
GeneMapper, PeakScanner or the online dnaLIMS FSA viewer is required to
view the files. Unfortunately the latter two programs do not allow
electropherograms to be superimposed to aid analysis. The Facility staff will
superimpose and analyze the electropherograms in consultation with the client,
and the resulting data will be sent to the client as images and peak data (relative
size, height, and area) in an excel file by email and/or .pdf files via dnaLIMS. In
addition, the Facility can generate images of the electropherograms with a JPEG
or GIF format to aid handling of the data by the client for papers, presentations,
etc.
4.3. Example of Results: In Figure 4a the electropherograms from a DNA Footprint
analysis or DNA protection assay were superimposed to show the protection area
as well as an example of a hyper sensitive site. The protection site is evident by
the blue peaks that are reduced in height compared to the red peaks in the area
defined by the brackets whereas the hypersensitive site is indicated by the three
peaks immediately to the left of the bracket in which the blue peaks are higher
than the red peaks. Otherwise the peak heights are fairly equal between the two
samples. The aligned sequence is evident in 2b which is read manually in this
case as the software does not have the capacity to interpret the base sequence
correctly.
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A.
B.
Figure 4. A) Electropherograms of the resulting DNA fragments from the DNA footprint
analysis. Blue = DNA plus protein of interest. Red = DNA plus bovine serum albumin.
B) DNA sequencing electropherograms superimposed and generated from the same
probe as in A and primed with the same oligonucleotide as in A. Red = T. Blue = C.
Green = A. Black = G.
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