Oligonucleotide-directed single-stranded DNA mutagenesis experiments
This paper describes a classic protocol consisting of the double primer technique of Zoller and Smith (1984,1987) combined with Kunkel's mutant yield enrichment method (1985). The single-stranded DNA templates used in this protocol contain high levels of uracil residues because they were prepared from M13 phages grown in dut and ung mutant E. coli (see Protocol 1). This experiment was derived from the next volume of the Laboratory Guide to Molecular Cloning (3rd edition) by [American] J. Sambrook D.W. Russell.
Operation method
Oligonucleotide-directed single-stranded DNA mutagenesis experiments
Materials and Instruments
E. coli strains suitable for transformation E. coli TG1 receptor for transformation Move makings For more product details, please visit Aladdin Scientific website.
ATP PE1 buffer PE2 buffer Phage T4 DNA ligase Phage T4 polynucleotide kinase E. coli DNA polymerase Ⅰ Klenow fragment M13 phage Universal sequencing primer dNTP solution containing 4 dNTPs Mutagenized M13 phage Single-stranded DNA template Mutagenized oligonucleotide primer Culture medium 2XYT top agar plate and YT agar plate
Falcon2059 Test tubes (pre-cooled) or shock cups 47°C Heating blocks or water baths 16°C, 42°C and 68°C water baths Water baths suitable for denaturation temperature
Buffers and solutions
Refer to Appendix 1 for various storage solutions, buffers and reagent compositions.
Dilute the stock solution to the desired concentration.
ATP (10 mmol/L)
10xPE1 buffer
200 mmol/L Tris-Cl (pH 7.5)
100 mmol/L MgCl2
500 mmol/L NaCl
10 mmol/L DTT
10XPE2 buffer
200 mmnl/L Tris-Cl (pH 7.5)
100 mmol/L MgCl2
100 mmol/L DTT
Enzyme and buffer
Phage T4DNA Ligase
Phage T4 Polynucleotide Kinase
E. coli DNA polymeraseⅠKlenow fragment
The extension reaction can be performed using any of the DNA polymerases (steps 4 and 5). The Klenow fragment lacks exonuclease activity at the 5' end and therefore cannot degrade the template. However, other enzymes such as phage T4DNA polymerase (Nossal 1974, Geisselsoder et al. 1987); the natural T7DNA polymerase (Bebenek and Kunkel 1989); and the sequencing enzyme Schena 1989, Venkitaiaman l989) are thin enough to require shorter incubation times. These enzymes must be used when phosphorylated mutagenic oligonucleotides are used as single primers for polymerization or extension reactions. Unlike the Klenow fragment, neither sequencing enzymes nor the natural DNA polymerases encoded by phage T4 and T7 can displace mutagenic oligonucleotides from the template (Nossal 1974, Kunkel 1985, Bebenek and Kunkel 1989, Schena 1989).
Nucleic acids and oligonucleotides
M13 Universal sequencing primers for phage
Any commercially available universal primer that uses the M13 single-stranded phage positive strand as a template for directing the DNA double deoxy sequencing reaction is suitable for use in this method.
A dNTP solution containing 4 dNTPs at 2 mmol/L each.
High-quality dNTP was used to minimize the chance of contaminating dUTPs being incorporated into newly synthesized strands of DNA. We believe that the concentrated dNTP solutions from Pharmacia work well.
Mutagenized M13 phage single-stranded DNA model wrenches
Mutagenized oligonucleotide primers
Mutagenic oligonucleotides should be designed as described in the Information Column on the topic of Mutagenic Oligonucleotides. Mutagenic oligonucleotides are purified by Sep-Pak C18 column chromatography to remove salt and other impurities prior to targeted mutagenesis (see Scheme 6 in Chapter 10). Purification of oligonucleotides by polyacrylamide gel electrophoresis is not required unless the cold nucleotides are longer than 30 clostridia or will be used for loop-in or loop-out mutagenesis.
Culture solution
2XYT top agar plates and YT agar plates
Specialized equipment
Falcon2059 test tubes (pre-cooled) or electroshock cups
47°C heating block or water bath
16°C, 42°C and 68°C water baths
Water baths suitable for denaturation temperatures.
See step 3, a thermal cycler can also be used in the above steps.
Other reagents
The reagents required for Step 1 are listed in Scheme 1 of this chapter.
The reagents required for Step 7 are listed in Schemes 3 and 4 in Chapter 12.
The reagents required for Step 7 are listed in Scheme 7 of this chapter.
Vectors and Strains
See Appendix 3
E. coli strains suitable for transformation (e.g. TG1)
E. coli TG1 receptor for transformation.
Receptor cell preparation is described in Chapter 1, Box 25 and 26.
E. coli strain TG1 overnight cultures
Methods
1. Prepare M13 phage single-stranded template as described in Scheme 1. Purify the uracil-containing template by centrifugal column chromatography.
2. Phosphorylate the mutagenic oligonucleotides and universal sequencing primers with phage T4 polynucleotide kinase by mixing the following in separate microcentrifuge tubes:
Synthesized oligonucleotides 100 to 200 pmoles
10X Phage T4 Polynucleotide Kinase Buffer 2ul
10 mmol/LATP 1ul
Phage T4 Polynucleotide Kinase 4 units
Add water to 20ul
Incubate at 37°C for 1 h, then heat at 68°C for 10 min to inactivate the polynucleotide kinase.
3. Complex phosphorylated mutagenic oligonucleotides and universal sequencing primers with single-stranded M13 phage DNA containing the target sequence. Mix the following reactants:
Single-stranded template DNA (-1ug) 0.5pmole
Phosphorylated mutagenic oligonucleotide 10pmoles
Phosphorylated universal primer 10pmoles
10XPF11 buffer 1ul
Add water to 10ul
Heat the above mixture for 5 min at a temperature more than 20°C above the theoretical Tm value for complete hybridization of the mutagenized oligonucleotide, and calculate the Tm value by the formula: Tm=4(G+C)+2(A+T), (G+C) is the sum of G and C residues in the oligonucleotide, and (A+T) is the sum of A and T residues in the oligonucleotide. Transfer the centrifuge tube containing the reaction mixture to a beaker containing water at a temperature more than 20°C above the Tm value. Place the beaker on a bench and allow it to cool slowly to room temperature (~20 min). Collect all condensate from the tube walls by slight centrifugation (5 s) in a microcentrifuge.
Alternatively, heating or cooling of nucleic acids and oligonucleotides can be performed in a thermal cycler. The molar concentration ratio of primer to template should be 10:1 or 50:1 Using too much primer will increase the frequency of ectopic mutations in the target DNA.
4. When the annealing reaction has cooled to room temperature, mix the following reagents in a new 0.5 ml microcentrifuge tube.
10 X PE2 Buffer 1.0ul
2 mmol/L dNTP solution 1.0ul
10 mmol/L ATP 1.0ul
Phage T4DNA Ligase 5Weiss Units
Klenow Fragment 2.5 units
Place the above mixture on ice and set aside.
When using phage T4DNA polymerase or sequencing enzymes, incubate the polymerization/extension reaction (step 5) at 0°C for 5 min, room temperature for 5 min, and then at 37°C for 2 h. Incubation at low temperatures facilitates synthesis starting from the 3' end of the DNA, and incubation at 37°C improves the efficiency of the extension reaction. In addition, the concentration of each of the four dNTPs in the reaction should be increased to 500 umol/L. This will significantly increase the efficiency of the extension reaction and strongly inhibit the 3' exonuclease activity of phage T4DNA polymerase.
5. Add 10ul of ice-cold Step 4 reaction mixture to the mixture containing single-stranded DNA and annealed oligonucleotides (Step 3). Incubate the final reaction mixture at 16°C for 6-15 h. Incubate the final reaction mixture for 2 h at 16°C for 2 hours.
6. Transfect suitable sensory E. coli host bacteria (e.g. TG1) as follows
a. Make serial dilutions (1:10, 1:100, 1:500) of the reaction mixture with 10 mmol/L Tris-Cl (pH 7.6). b. Incubate the final reaction mixture at 16°C for 6 to 15 h.
b. Prepare a series of pre-cooled (0°C) Falcon 2059 centrifuge tubes by transferring (i) 1ul, 5ul of the original reaction mixture and (ii) 1ul, 5ul of various dilutions of the reaction mixture into the tubes respectively. 200ul of Receptorized TG1 Cell Preparation was added to each centrifuge tube.
c. Place the above mixture on ice for 30 min, then transfer to a 42°C water bath and hold for exactly 2 min.
d. Remove the reaction from the 42°C water bath and add 100ul of TGl to culture the cells overnight. The addition of excess cells makes it easier to observe the M13 phage spots growing in the moss.
If freshly prepared TG1 cells are used in step b instead of frozen sensory cells, no additional TG1 cells need to be added.
e. Add 2.5 ml of 2xYT Top Agar (melted and cooled to 47°C) to each centrifuge tube, mix, and spread on the top layer of the YT Agar plate. Incubate the plate at 37°C for 12-16 h to allow for the formation of phage spots.
Mutant DNA can also be introduced into E. coli by electroshock. The frequency of transfection by electroshock is usually much higher than that of normal transfection, so before electroshocking, each portion of the mutagenic reaction mixture should be serially diluted with water 1/100; 1/500; 1/1000; and 1/10,000 times. 1~10ul of the diluted solution should be taken for electroshocking. 
7. Screen the phage plaque by sequencing of single-stranded phage DNA (see Scenarios 3 and 4 in Chapter 12). If required, screen for low-frequency mutants by radioisotope-labeled oligonucleotide probes. 