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| The proportion of transformants that carry the mutated sequence is higher in this protocol than in traditional site-directed mutagenesis protocols. This is because the uracil-containing wild-type DNA is degraded in the ung+ strain, leaving the majority of the transformants derived from the uracil-free strand primed by the mutagenic oligonucleotide. T4 DNA polymerase has a very high fidelity and therefore you only need to sequence across the oligonucleotide junctions |
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A. Template Preparation
1. Introduce a recombinant phage, such as M13, or plasmid with a single stranded origin of replication containing the template DNA of interest into the E. coli strain CJ236 (F', ung-, dut-) (see Hint #1).
2. Innoculate 2X YT media containing 0.25 μg/ml Uridine with a one-tenth volume mid-log phase culture of the recombinant strain. Incubate at 37°C with vigorous shaking for 6 hr.
3. Prepare uracil-containing, single-stranded DNA (see Protocol on Preparation of Single-Stranded DNA for Site-Directed Mutagenesis and see Hint #2)
B. Phosphorylation of Mutagenic Oligonucleotide Primer
1. Dissolve desiccated oligonucleotide and quantify the solution by UV spectrophotometry (see Hint #3).
2. Set up the following kinase reaction:
2.5 μg of Oligonucleotide
5 μl of 10X PNK Buffer
5 Units of T4 Polynucleotide Kinase
5 μl of 10 mM ATP
Add ddH2O to a final reaction volume of 50 μl
3. Incubate the reaction for 15 min at 30°C and then keep it on ice until its use.
C. Annealing Reaction (see Hint #4 and #5)
1. Set up the following annealing reaction:
50 ng of Phosphorylated Oligonucleotide (from Section B)
100 ng of ssDNA template (from Section A)
1 μl of 10X Annealing Buffer
Add ddH2O to a final reaction volume of 10 μl
2. Incubate the reaction in a beaker containing 70°C ddH2O and allow the beaker contents to cool to room temperature (see Hint #6 and #7).
D. Synthesis Reaction
1. Set up the following synthesis reaction
10 μl of Annealing Reaction Solution
1 μl of 10X Synthesis Buffer
3 Units of T4 DNA Ligase
4 Units of T4 DNA Polymerase
2. Incubate the reaction for 5 min on ice.
3. Incubate the reaction for 5 min at room temperature.
4. Incubate the reaction for 90 min at 37°C (see Hint #8).
5. Take 7 μl of the reaction mix and transform into MV1190 or DH5-α (ung-) competent cells (see Hint #9).
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| PNK Buffer (10X) |
| 50 mM DTT
10 mM Spermidine
100 mM MgCl2
700 mM Tris-HCl, pH 7.6
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| 10 mM ATP |
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| Synthesis Buffer (10X) |
| 5 mM dCTP
5 mM dGTP
5 mM dATP
10 mM ATP
5 mM dTTP
20 mM DTT
50 mM MgCl2
100 mM Tris-HCl, pH 7.4
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| Annealing Buffer (10X) |
| 500 mM NaCl
20 mM MgCl2
200 mM Tris-HCl, pH 7.4
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| 2X YT Medium |
| 16 g/liter Tryptone
10 g/liter Yeast Extract
Autoclave
86 mM NaCl
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Sodium Chloride
Uridine
Yeast Extract
Tryptone
Spermidine
DNA Ligase, T4
Magnesium Chloride
Oligonucleotide
Tris-HCl
dCTP
ATP
dTTP
dGTP
dATP
Polynucleotide Kinase, T4
DNA Polymerase, T4
DTT
E. coli strain CJ236 (ung-, dut-, F')
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1. In general, it is a good idea that the target DNA be less than 1 kb. Large stretches of DNA are unstable in single-stranded phage vectors. Also, a large target DNA increases the chances that the mutagenic primer will anneal to the wrong sequence. Finally, at the conclusion of the mutagenesis, the target DNA must be sequenced to determine that only the desired mutation is present. A shorter fragment is easier and cheaper to sequence.
2. To test uracil incorporation into the single-stranded DNA, check that the single-stranded DNA can transform a dut- ung- strain efficiently but not an ung+ strain. Use 10 ng of single-stranded DNA for the transformation.
3. One unit of absorbance at 260 nm is approximately 30 μg/ml.
4. Include a negative control reaction without the Oligonucleotide and carry it though all of the following steps. Upon transformation, this reaction should yield no or few transformants.
5. Difference sources report different ratios of phosphorylated mutagenic oligonucleotide to single-stranded template DNA ranging from 2-fold molar excess to 30-fold excess.
6. Alternatively, a thermal cycler could be used to bring down the temperature from 70°C to 25°C in 1°C decrements every min and then cool to 0°C.
7. The annealing temperature should be at Tm +20°C. To calculate the theoretical Tm (assuming a perfect match between the oligonucleotide and the single-stranded DNA), use the formula Tm = 4(G+C) +2(A+T).
8. The room temperature incubation optimizes initiation from the 3' termini while the 37°C incubation favors the extension reaction.
9. Check the colonies by hybridization and then sequence the positive clones to ensure that only the desired mutation is present in the DNA.
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2. Kunkel TA, Roberts, JD, Zakour, RA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987; 155:166.
1. Kunkel TA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. 1985; 82:448.
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