Permanganate genomic footprinting salivary glands (Modified 6/26/06).

Lab protocol: I have also found that the analysis works well with as little as 50 ng of Drosophila DNA. I add 150 ng of sonicated salmon DNA as a carrier. This can be considered as an alternative to the 100 ng of Drosophila DNA that I describe in the protocol.  Some students have found that using 150 to 200 ng of DNA gives more consistant results.

You can use Potassium Permanganate on living cells in order to detect the presence of RNA polymerase II on the hsp70 promoter.  Permanganate reacts preferentially with thymines located in single-stranded regions of DNA.  This detects the transcription bubble associated with the paused polymerase found on the hsp70 promoter.  There is also a low level of reactivity with thymines situated elsewhere in the promoter.  It turns out that the thymines situated in the TATA box are weakly protected from permanganate by TFIID.

Permanganate, like another widely used footprinting reagent Dimethyl Sulfate, does not actually cleave the DNA.  It oxidizes the C5-C6 double bond in thymines and renders the DNA backbone sensitive to cleavage by piperidine.  Importantly, the products generated by the piperidine cleavage reaction carry a 5' phosphate at the point of cleavage and this cleavage can be detected by ligation-mediated PCR.


Dissection of salivary glands and permanganate treatment.
Before starting, prepare a fresh stock of 40 mM KMnO4 and KMnO4 stop solution. The 40 mM KMnO4 is prepared in dissection buffer (130 mM NaCl, 5 mM KCl, 1.5 mM CaCl2) and stored on ice. KMnO4 stop solution consists of 20 mM Tris pH 7.5, 20 mM NaCl, 40 mM EDTA, 1% SDS, 0.4M ß-MeOH (note that the stock solution of ß-MeOH is 14.3 M). Keep the stop solution at room temperature so the SDS doesn't precipitate.
40 mM KMnO4 (100 ul/rxn)

1 ml dissection buffer 

6.32 mg KMnO4

Rock at room temp. for 10' and store on ice.  This is very important because the dark color of the solution makes it difficult to see when the crystals have dissolved.

 MnO4 stop solution (200ul/rxn)

100 ul 1 M Tris-Cl pH 7.5 

20 ul 5 M NaCl 

400 ul 0.5 M Na2EDTA pH 8 

500 ul 10% SDS 

280 ul ß-MeOH (this has been doubled from the original protocol to insure complete reduction of the permanganate)

3.7 ml H2O
Siliconize enough 1.7 ml tubes for the number of samples that you will generate. Each sample will consist of approximately 12 glands. Siliconize tubes by adding a drop of Sigmacote to each tube and splashing the solution across the tube surface. Pour the residual Sigmacote into a waste beaker in the hood and set the tubes aside to air dry. After they have dried, rinse each with water.

Six or more larvae are placed on moist filter paper in a small petri dish. As they crawl around, debris attached to the larvae should transfer onto the filter paper. If the larvae are to be heat shocked, three petri dishes with moist paper are preheated in the 37°C incubator. At 5 minute intervals, 2 to 4 larvae are placed in a dish. They are heat shocked for 15 to 20 minutes and then dissected. By setting up three dishes, it is possible to dissect 6 larvae while keeping the heat shocks within the 15 to 20 minute period.

Put 100 ul of dissection buffer (130 mM NaCl, 5 mM KCl, 1.5 mM CaCl2) in a siliconized 1.7 ml tube and place on ice.

Place one larva in a well of the glass slide containing approximately 100 ul of dissection buffer. Dissect out the salivary glands with a pair of fine-tipped forceps and carefully transfer the glands with forceps to the 100 ul of dissection buffer in the siliconized tube stored on ice. Continue dissections until 12 glands have been transferred to the siliconized tube. This will take about 20 minutes. You should permanganate treat a collection of glands before going onto another collection of glands so that the glands are not left for for an unnecessarily long length of time. As you are collecting the salivary glands in one siliconized tube, also transfer soft body parts from each larva to another siliconized tube. DNA will be isolated from these soft body parts in parallel with the salivary glands. This DNA serves for making the "naked DNA" control by reacting it with permanganate. It also serves for making G/A markers.

Add 100 ul of 40 mM permanganate to the tube of salivary glands and gently tap the sides of the tube to promote mixing. Avoid vigorous mixing as the glands can stick to the sides of the tube above the permanganate solution. Put the sample back on ice for 2 minutes.

After 2 minutes, add 200 ul of stop solution to both the salivary glands and the soft body tissue. Tap the sides of the tube to mix. The solution should quickly change from purple to clear. The glands will remain a brownish color for about 10 seconds. At this point, add 100 ul of dissection buffer to the lysed soft body parts so the volume of this sample is comparable to the volume of the permanganate treated glands. Place the samples in the Brinkmann shaker for for at least 10 minutes and procede with additional dissections if desired.

Once all the permaganate reactions are completed, add 50 ug of proteinase K to each sample. Digest for one hour at 37°C.

Extract the sample with a sequence of equilibrated phenol (pH 8), equilibrated phenol (pH 8), and chloroform. Each extraction is done with 300 ul of the organic solution. Samples are shaken for 5 minutes and centrifuged for 5 minutes to separate phases. For both of the phenol extractions, the aqueous phase is transferred to a fresh unsiliconized tube. For the chloroform extraction, the chloroform layer is removed by pipetting it out from underneath the aqueous phase.

Add 40 ul of 3M Na acetate pH 7 and 1 ml of cold Ethanol. Thoroughly mix by inverting the tube and place on ice for at least 15 minutes. Microfuge in the cold for at least 20 minutes. A small pellet should be evident. Discard the supernatant and add 100 ul of cold 75% Ethanol. Microfuge in the cold for 5 minutes and discard the supernatant. Allow the samples to air-dry.

Dissolve the nucleic acid pellet from the salivary glands in 20 ul of TE (10 mM Tris pH 7.5, 1 mM EDTA), and the nuclei acid pellet from the soft body parts in 50 ul of TE.

Determine the concentration of DNA by measuring 1 ul of the sample on the Fluorometer. DNA concentrations for samples derived from 12 glands are typically around 10 ng/ul and around 50 ng/ul for the the corresponding soft body parts. Note, that the samples contain RNA, and this probably accounts for why a pellect can be seen in the previous ethanol precipitation. The RNA will not interfere with the Fluorometer measurements, nor with the LM-PCR.

Store samples in the refrigerator for short term or in the freezer for long term.


Permanganate treatment of naked DNA.

In order to interpret the pattern of permanganate reactivity that occurs in in vivo, the pattern must be compared to that which occurs in naked genomic DNA.

Prepare one tube containing 300 ng of the genomic DNA purified from soft body tissue or salivary glands and diluted to a final volume of 100 ul in dissection buffer. Place on ice.

Add 100 ul of ice cold 40 mM KMnO4 (dissolved in dissection buffer), and incubate for 60 seconds. This level of treatment for naked DNA matches well with the 2' treatment of intact salivary glands.

Stop the reaction with 200 ul of MnO4 stop solution.

Add 40 ul of 3M Na acetate pH 7 and 1 ml of Ethanol. Ice for 15', microfuge for 20', discard supernatant and wash pellet with cold 75% ethanol. Note that this DNA is not phenol extracted since it should already be protein-free.

Air dry the DNA and then dissolve it in 20 ul of TE. Quantify the DNA with a fluorometer.


Restriction enzyme digestion of genomic DNA.

Although this is not normally done, there may be situations were you want a parental band in order to emphasize that comparable amounts of material have been analyzed. Note, however, that some combinations of samples such as different 5' deletions will generate parentals of different sizes. This will probably detract from the data.

In a siliconized tube, digest 100 ng of DNA in a total volume of 25 ul with the desired enzyme.

Add 2.5 ul of 3M Na acetate and 62.5 ul of Ethanol. Place on ice for 15' and precipitate DNA as described above. Include a 75% Ethanol wash. Allow the samples to air dry and then dissolve in 15 ul of TE.


Piperidine cleavage of the permanganate-treated DNA.

Piperidine cleaves the DNA at the oxidized thymine residues. One hundred nanograms of DNA should start in a total volume of 15 ul of TE. The DNA is put in a siliconized 0.65 ml tubes. Add 75 ul of H2O and 10 ul of piperidine. Heat the samples at 90°C for 30 minutes in the thermocycler.

After 30 minutes, add 300 ul of water to the DNA solution and transfer each sample to a 1.7 ml tube.  Isobutanol extract 2 times using 800 ul each time and 1 time using 400 ul of isobutanol.  Isobutanol will be the top phase and should be discarded.  Extract one time with ether.  Adjust the volume of DNA to 100 ul by adding water.  Ethanol precipitate the DNA using 10 ul of 3M Na acetate pH 7.0 and 250 ul of ethanol.  Precipitate the DNA and wash with 75% ethanol in the routine way.  Dissolve permanganate samples in 10 ul of 1/2 X TE.   Transfer the DNA to a fresh siliconized 600ul tube. This transfer is essential since there is something about the tube where the piperidine reaction was done that completely inhibits the Vent polymerase. Its now ready for LM-PCR.


Generation of G/A markers using depurination with formic acid.

Formic acid depurinates the DNA and subsequent cooking with piperidine cleaves the DNA backbone. Formic acid treating 1 ug of DNA should yeild enough material for 10 gels.

In a siliconized 1.7 ml tube, combine 1 ug of genomic DNA in 10 ul of TE with 10 ul of H2O and 50 ul of 99% formic acid. Incubate at 15°C for 5'.

Add 200 ul of cold 0.3 M Na acetate pH 7.0, 50 ug/ml tRNA to stop depurination. Add 750 ul of cold Ethanol and ice for 15 minutes. Microfuge in the cold for 15 minutes to collect the DNA. Wash pellet with 200 ul of 75% cold Ethanol and air dry.

Dissolve the DNA in 90 ul of H2O and transfer to a siliconized 600 ul tube. Add 10 ul of piperidine and incubate at 90°C for 30 minutes.

Remove the piperidine as described above for piperidine cleavage of permanganate-treated DNA.  Air dry the final ethanol precipitate and dissolve in 50 ul of 1/2X TE.

By subjecting 10 ul of the DNA to the LM-PCR protocol, enough radiolabelled DNA will be made to easily detect a G/A ladder on at least two gels with overnight exposures.


LM-PCR analysis. (Note that an updated version of this protocol presented in the form of a worksheet by going here - there are some volume changes to streamline the protocol.)

The protocol is derived from Garrity and Wold's procedure found in Current Protocols. Slight modifications were introduced by Jan Weber, and the current description is specific for the set of primers that anneal to ß-gal sequences downstream from the transformed promoters. These primers are called TR-1, TR-2, and TR-3. Obviously, if you intend to use a different set of primers, you will probably have to adjust the PCR conditions. Some examples can be found here.

Be sure to mix all solutions thoroughly using gentle triteration. To improve accuracy, prepare master mixes of various solutions. Prepare the master mixes by multiplying the amount of material for one reaction by the sum of 0.5 and the number of samples being processed. This ensures that there will be enough master mix for all of the samples. Also, check the PCR programs to make sure they are correct - sometimes they get modified and not returned to their original state.

Combine 10 ul of DNA (100 ng in TE) with 20 ul of First strand synthesis mix.  In the thermocycler, incubate at 95°C for 5 minutes, 50°C for 30 minutes, and 76°C for 10 minutes. Spin down condensate and place on ice.

First strand synthesis mix (20ul/rxn)

12.75 ul H2O 

6ul 5X First Strand Buffer* (heat briefly to 37oC to ensure that the geletin is fully dissolved)

0.5 ul of 1 pmoles/ul TR-1

0.5 ul 10 mM dNTPs 

0.25 ul 2U/ul Vent polymerase (NEB #254L)

Shortly before use, premix  17.5 ul of Modified Ligation Dilution Solution (MLDS)* and 2.5 ul of 1 mg/ml BSA per  sample; then add this mix to the sample. 

Next, add 25 ul of Ligation Mix. Incubate overnight at 15°C.
Ligation Mix (25ul/rxn)

9.25 ul Modified Ligation Premix*

7.5 ul 10 mM rATP 

1.25 ul 1mg/ml BSA 

5 ul Linker pair (A':B at 20 pmoles/ul) 

2 ul T4 DNA ligase (1Weiss Unit/ul)
*11/13/96: I reduced the ligase from 3U (original protocol) to 2U and added 1ul of water to make-up the difference. Strong signals were still obtained. Add 9.4 ul of salt precipitation mix* and 220 ul cold Ethanol. Mix well and place on ice for at least 15 minutes. Microfuge in cold for 20 minutes. Wash one time with 100 ul of 75% ethanol, microfuging to secure the precipitate. Air dry the pellet.
Dissolve the DNA precipitates in 30 ul of H2O.  Place samples on ice.  Add 70 ul of Vent amplification buffer.
Vent Amplification buffer (70ul/rxn)

20 ul 5X Vent Amplification Buffer* (heat briefly to 37oC to ensure that the geletin is fully dissolved)

1 ul of 10 pmole/ul TR-2 

1 ul of 10 pmole/ul Linker primer A' 

2 ul 10 mM dNTP 

0.5 ul Vent polymerase 

45.5 ul H2O

Perform 18 amplification cycles. (This can be altered if your signals are weak.  I have gone up to 20 cycles.) The first cycle should have a 3 minute denaturing step at 95°C and the remaining should be for 1 minute. All cycles have annealling steps of 2 minutes. For TR-2, this is 58°C. The first 9 cycles have extension steps of 3 minutes and the last 9 cycles have extension steps of 4 minutes. All extension steps are at 76°C. The two sets of 9 cycles is provided by program 2. Following amplification, the tubes are placed on ice.

If it is not already done, radiolabel TR-3 with kinase. The following reaction will give you enough primer to label 20 samples if you adjust the final volume of the primer so that it is a little more than 100 ul. Combine 6.3 ul of H2O, 1 ul of 10X kinase buffer, 0.4 ul of 0.1 mM TR-3, 2 ul 32P-g ATP, and 0.3 ul kinase. Incubate at 37°C for 30 minutes. We used to purify the oligonucleotide on a Nensorb column. As of September 1997, we have been purifying radiolabelled oligonucleotides on Stratagene's NucTrap columns. Since the oligonucleotide is recovered in a aqueous solution (rather than 50% methanol) it can be used straight from the NucTrap column. You should obtain at least 106 cpm/pmole. As of 2005, we have been purifying radiolabelled oligonucleotides with spin columns from eith BioRad or Amersham.

After the 18 cycles of amplification, proceed with the labelling reaction. Add 20 ul of labelling mix to each sample. Perform two amplification cycles using program 3. The first cycle is initiated with a 3 minute denaturation at 95°C and the second cycle is for 1 minute. Annealling is done for 2 minutes at 62°C. Extensions are done for 10 minutes at 76°C. Following the labelling cycles, hold tubes at room temperature.

Labelling mix (20ul/rxn *)

9.5 ul H2O 

4 ul 5X Vent Amplification Buffer* (see above) 

1 ul 10 mM dNTP 

5 ul radiolabelled TR-3 (approximately 2 pmoles) 

0.5 ul Vent Polymerase
*11/13/96: In my last experiment, I realized that I was going to run out of mix. I added 10ul rather than 20ul to the last two samples and then dissolved the resulting samples in 6ul rather than 12ul of sequencing loading buffer. The samples were well matched on the gel indicating that reducing the labelling mix by 2 results a proportional decrease in labelled material. There was still plenty for the gel.

At the end of the labelling reaction, add 14 ul of Stop solution*.  Transfer 120 ul of each reaction to a fresh 1.7 ml tube (the larger tube facilitates the organic extraction).  Extract one time with 100 ul of Phenol:Chloroform:Isoamyl Alcohol (25:24:1) {Caution, highly radioactive!). Transfer 100 ul of the aqueous phase to a fresh 1.7 ml tube and add 250 ul of cold Ethanol. Chill on ice for 15 minutes and then microfuge for 15 minutes. Remove supernatant and wash pellet with 100 ul of 75% ethanol, microfuge and discard supernatant. Air dry the samples.

Dissolve precipitates in 10 to 15 ul of sequencing loading buffer. For gel analysis, boil 3-5 ul and reserve the rest for a subsequent gel if this is needed.


Recipe for 8% sequencing gel mix (the following is enough for 5 gels @ 30ml/gel).  This stock is stored at room temperature and is good for only a few weeks.

Dissolve the following in water to give a total volume of 157.5 ml:

84 g Urea

13.3 g  acrylamide

0.7 g  bis-acrylamide
Add 17.5 ml of 10X TBE. This results in a final volume of 175 ml of 8% gel mix.

For our sequencing plates, mix 35 ml of sequencing gel mix, 200 ul of 10% ammonium persulfate, and 23 ul of TEMED.  Allow the gel to polymerize for at least 1 hour. 
LMPCR Stock solutions:
5X First Strand Buffer (Store in freezer, warm to 37oC before use to thoroughly dissolve gelatin) Modified Ligation Dilution Solution (MLDS, store in freezer) Modified Ligation Premix (Store in freezer) Salt Precipitation Mix (Store in freezer) 5X Vent Amplification Buffer (Store in freezer, warm to 37oC before use to thoroughly dissolve gelatin) Stop Solution (Store in freezer) Preparation of A':B linker pair
In a 1.5 ml microfuge tube, prepare 1 ml of solution containing final concentrations of 250 mM Tris-Cl pH 7.7, 20 uM Linker A' and 20 uM Linker B.  Heat the solution to 95oC for 5 minutes and then float the tube in a beaker containing 250 ml of water adjusted to 70oC.  Allow the water to cool to room temperature and then leave the tubes at room temperature for at least one more hour.  The beaker of water containing the tube of oligonucleotides is then place on ice so that it slowly cools to 4oC and then the tubes are left on ice overnight.  Store the oligonucleotide solution at -20oC.  Don't warm the solution above room temperature when you thaw the tubes.

Alternatively, the oligonucleotides can be annealed in the PCR machine.  Divide the solution above into 5 x 200 ul portions in PCR tubes.  Subject to the following: 95oC for 5', 70oC for 5', 65oC for 5' , continue decreasing 5oC increments and incubating 5' until the samples reach 20oC, 20oC for 60', 18oC for 5', 16oC for 5', 14oC for 5', 12oC for 5', 10oC until its convenient to put at 4oC overnight.  Finally, store in the freezer.