DNase I Footprinting

互联网2013-09-06

1532

Overview

An end labeled DNA probe is incubated with a purified DNA-binding factor or with a protein extract. The unprotected DNA is then digested with DNase I such that on average, every DNA molecule is cut once. Digestion products are then resolved by electrophoresis. Comparison of the DNase I digestion pattern in the presence or absence of protein will allow the identification of a footprint (protected region).

Procedure

1. Make the DNA binding reaction by combining the following components in a microcentrifuge tube:

25 μl of protein extract in HEMG

10 μl of 10% Polyvinyl Alcohol

1 μl of 1 M HEPES, pH 7.6

2 fmol of end-labeled DNA probe (about 1200 cpm)

1 to 5 μg competitor DNA

and bring the final reaction volume to 50 μl with ddH₂ O.

2. Mix the components gently and incubate on ice for 10 min (for incubations with purified proteins, incubation temperatures may be increased).

3. Add 50 μl of Ca/Mg Solution to the binding reaction at room temperature.

4. Add 1 to 10 μl of DNase I solution (see Hint #1).

5. Mix quickly and incubate at room temperature for 1 min.

6. Stop the reaction by adding 100 μl of stop solution and mix by vortexing immediately.

7. Add 200 μl of phenol/chloroform and mix by inverting several times. Centrifuge at full speed in a microcentrifuge for 10 min to separate the phases. Recover the aqueous phase and transfer it to a fresh microcentrifuge tube.

8. Add 1 ml of 100% ethanol and mix by inverting several times.

9. Centrifuge at full speed in a microcentrifuge for 10 min to pellet the DNA and remove the ethanol.

10. Resuspend the pellet in 70% ethanol and pellet the DNA again. Remove the ethanol and allow the DNA pellet to air dry.

11. Resuspend the DNA in 6 μl formamide dye and load the sample onto a 6% to 8% sequencing gel (see Protocol on Sequencing Gel Protocol).

Solutions

Competitor DNA		Sonicated Calf Thymus DNA in ddH₂ O

Formamide Dye		0.005% (w/v) Xylene Cyanol FF 20 mM EDTA make up in deionized Formamide 0.005% (w/v) Bromophenol Blue

70% (v/v) Ethanol

1:1 Phenol:Chloroform

Stop Solution		1% (w/v) SDS 0.2 M NaCl Store at room temperature 20 mM EDTA, pH 8.0 0.25 mg/ml carrier RNA

Ca/Mg Solution		5 mM CaCl₂ 10 mM MgCl₂

DNase I		2.5 mg/ml in ddH₂ O Freeze as 2-5 μl aliquots and store at -70°C

HEMG		0.1 mM EDTA, pH 8.0 0.1 M KCl 25 mM HEPES, pH 7.6 with KOH 12.5 mM MgCl₂ 10% (v/v) Glycerol 1 mM DTT (add just before use)

1 M HEPES		pH 7.6 with KOH

10% (v/v) Polyvinyl Alcohol

BioReagents and Chemicals

SDS
EDTA
DNase I
HEPES
Polyvinyl Alcohol
Calcium Chloride
Sonicated Calf Thymus DNA
Ethanol
Magnesium Chloride
Chloroform
Phenol
Glycerol
Formamide
Potassium Hydroxide
DTT
Potassium Chloride
Oligonucleotide
Sodium Chloride
Xylene Cyanol FF
Bromophenol Blue
carrier RNA

Protocol Hints

1. Make a fresh dilution of the DNase I stock solution in ice cold ddH₂ O and keep on ice. For the no-protein control, use about 1 μl of a 1:1000 dilution. For crude protein extracts, the amount and dilution of the DNase I needs to be determined empirically; 5 μl of a 1:100 dilution is usually adequate.