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| The protocol describes how to make a series of deletions in a cloned gene of interest using Exonuclease III. The plasmid containing the gene of interest is digested with enzymes that cut downstream of the target DNA. The site closest to the target DNA is cut with an enzyme that leaves a blunt end or a 5' overhang. The other site is cut with an enzyme that leaves a 3' overhang. Because Exonuclease III is able to digest from a 5' overhand end or a blunt end, and a 3' overhang is resistant to the enzyme's activity, deletions occur only on one end of the insert. In this protocol, the digestion by Exo III is prevented from digesting one end of the insert by digesting with a restriction enzyme that leaves a 5' overhang and then filling in with thio-dNTP's. Exo III's rate of exonucleolytic activity is very uniform; thus, incubations for differing lengths of time with the enzyme will yield different endpoints for the deletions. After the Exo III digestion, the DNA is treated with Mung Bean Nuclease to remove the remaining single strands and the DNA is religated together. |
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1. Set up a restriction digest with a restriction enzyme that creates a 5' overhang on the site to be protected from deletion (see Hints #1, #2, #3, and #4). This end will be filled-in in Steps #5 and #6 to prevent Exo III action. Alternatively, use a restriction enzyme that creates a 3' overhang and skip Steps #5 through #7. Use 2.5 μg of DNA per Exo III incubation time point.
2. Incubate for at least 1 hr at the temperature suggested for the restriction enzyme chosen.
3. Incubate restriction digest at 70°C for 15 min to heat inactivate the enzyme.
4. Place the tube on ice.
5. Add Thio-dNTPs to 40 μM and add 5 Units of Klenow fragment.
6. Incubate 20 min at room temperature.
7. Heat the samples at 65°C for 10 min to inactivate the enzyme.
8. Phenol/Chloroform extract the DNA by adding an equal volume of Phenol:Chloroform:Isoamyl Alcohol and vortexing to mix.
9. Centrifuge for 3 min at maximum speed in a microcentrifuge to separate the phases.
10. Collect the upper, aqueous phase.
11. Precipitate the DNA by adding 0.1 volume of 3 M Sodium Acetate and 2 volumes of Ethanol.
12. Incubate at -20°C, if desired.
13. Centrifuge for 5 min at maximum speed in a microcentrifuge to pellet the DNA.
14. Set up a restriction digest with the second Restriction Enzyme on the side of the target DNA from which deletions are to proceed in a volume of approximately 100 μl. Use a restriction enzyme that leaves a blunt end or a 5' overhang (e.g. Eco RV or EcoR1, respectively) (see Hint #1).
15. Incubate for at least 1 hr at the temperature suggested for the restriction enzyme chosen.
16. Heat inactivate the enzyme when the reaction is complete by heating the extract to 65°C for 10 min.
17. Dispense 20 μl of 10X Mung Bean Nuclease Buffer and 155 μl of ddH2O into microcentrifuge tubes. Set up enough for each time point. Prepare a container of Dry Ice.
18. Resuspend the DNA in 1X Exo Buffer and 2-Mercaptoethanol to a final concentration of 10 mM. Add the appropriate amount of ddH2O for a final volume of 25 μl per time point.
19. Incubate at 30°C for 5 min to equilibrate.
20. Add 20 Units ExoIII per μg of DNA and vortex to mix.
21. Incubate according to the following formulae for ExoIII digestion: 37°C = 400 bp/min, 34°C = 375 bp/min, 30°C = 230 bp/min, 23°C = 125 bp/min.
22. Remove a 25 μl aliquot at an initial time point and thereafter at 40 to 80 sec intervals. Generally use an incubation temperature of 30°C and take the first aliquot at 20 sec.
23. Place the aliquots into the tubes containing the diluted Mung Bean Nuclease Buffer. Immediately place on Dry Ice.
24. When the incubations are finished, remove the aliquots from the Dry Ice and heat at 68°C for 15 min. Place on wet ice.
25. To make the DNA blunt-ended, add to each aliquot 1 μl of Mung Bean Nuclease Solution per 2.5 μg DNA.
26. Incubate 30°C for 30 min.
27. Ethanol precipitate by adding 0.1 volume of 3 M Sodium Acetate and 2 volumes of Ethanol.
28. Centrifuge for 5 min at maximum speed in a microcentrifuge.
29. Resuspend the DNA pellet in 6 μl of TE.
30. Add 1 μl of DNA Loading Buffer and load the samples onto a 0.8% Agarose Gel made up in 1X TAE. A linearized clone and linearized vector (1 μg each) provide useful markers for the start and end of the deletion series, respectively. Run the gel.
31. Excise the bands of interest and isolate the DNA from the gel (see Protocol on Isolation of DNA from Agarose Gels and see Hint #5).
32. Set up an overnight ligation to recircularize the digested plasmids (see Protocol on DNA Ligation).
33. Transform competent bacteria with the ligation mix and pick 2 to 4 colonies per time point for mini-gel analysis and subsequent sequencing (see Protocol on Bacterial Transformation).
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| TAE Buffer (50X) |
| 57.1 ml/liter Glacial Acetic Acid
Working concentrations: 40 mM Tris-acetate, 2 mM EDTA
37.2 g/liter Disodium EDTA
Adjust pH to 8.5
242 g/liter Tris Base
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| DNA Loading Buffer (6X) |
| 0.25% (w/v) Bromophenol Blue
0.25% (w/v) Xylene Cyanol
30% (v/v) Glycerol
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| TE |
| 10 mM Tris
pH 8.0
1 mM EDTA
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| 3 M Sodium Acetate |
| 3 M Sodium Acetate, pH 5.2
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| Mung Bean Nuclease Dilution Buffer |
| 10 mM Sodium Acetate, pH 5.0
50% (v/v) Glycerol
0.1 mM ZnCl
0.1% (v/v) Triton X-100
1 mM Cysteine
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| Mung Bean Nuclease Solution |
| 7.5 Units Mung Bean Nuclease
Prepare in Mung Bean Nuclease Dilution Buffer
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| Mung Bean Buffer (10X) |
| 500 mM NaCl
50% (v/v) Glycerol
10 mM ZnCl
300 mM Sodium Acetate, pH 5.0
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| Exo Buffer (2X) |
| 100 mM Tris, pH 8.0
20 μg/ml tRNA
10 mM MgCl2
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| Phenol:Chloroform:Isoamyl Alcohol |
| Store at 4°C in a dark glass bottle
25:24:1 Tris-equilibrated Phenol:Chloroform:Isoamyl Alcohol (CAUTION! see Hint #6)
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| Thio-dNTPs |
| 0.4 mM Thio-dCTP
0.4 mM Thio-dTTP
0.4 mM Thio-dGTP
0.4 mM Thio-dATP
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Cysteine
Triton X-100
Glycerol
Dry Ice
EDTA
Tris Base
Magnesium Chloride
Isoamyl Alcohol
Chloroform
tRNA
Phenol
Restriction Enzyme
Ethanol
Zinc Chloride
2-Mercaptoethanol
Sodium Chloride
Klenow Fragment
Mung Bean Nuclease
Glacial Acetic Acid
Thio-dCTP
Thio-dTTP
Thio-dGTP
Thio-dATP
Sodium Acetate
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1. ExoIII has a 3' to 5' exonuclease activity that initiates from a blunt ended DNA or from a 5' overhang.
2. The DNA must be >85% supercoiled. ExoIII will digest from nicks. Use Cesium Chloride-purified DNA and AVOID USING UV to isolate the DNA. Check the condition of the plasmid DNA on a gel and compare to digested plasmid.
3. To prevent deletions in the both directions, cut one side with an enzyme that leaves a 3' overhang (e.g. Kpn, NOT SacII, which won't work) or cut with a Restriction Enzyme that leaves a 5' overhang and fill in with thionucleiotides as described in Steps #5 and #6. The contributors of this protocol believe this is more reliable than using a Restriction Enzyme that create a 3' overhang.
4. The DNA must be fully restricted with the 5' overhang Restriction Enzyme (otherwise you will see a background of undigested DNA) and with the 3' overhang Restriction Enzyme (otherwise deletions will proceed in both directions).
5. Use a gel to isolate the deletion fragments rather than simply extracting the DNA. (Extractions of the deletions after the Mung Bean Nuclease step requires the addition of 4 μl of 20% SDS, 10 μl of 1 M Tris, pH 9.5, 20 μl of 8 M Lithium Chloride, and 250 μl of Phenol:Chloroform, followed by re-extraction and precipitation. Gel purification is preferable because it removes the background and allows you to monitor how well the reaction has proceeded.)
6. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
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