In vitro mutagenesis using double-stranded DNA as a template: mutant selection experiment with DpnⅠ
In vitro mutagenesis using double-stranded DNA as a template: mutant selection experiment with DpnⅠ
This scheme and Scheme 4 use two oligonucleotides and high-fidelity polymerase to guide DNA synthesis using denatured plasmid DNA as a template. In this scheme, full-length double-stranded plasmid DNA will be amplified in linear form after multiple rounds of thermal cycling to produce a mutant plasmid with a staggered nick in the DNA double strand (Hemsleyetal.1989). This experiment was derived from the next volume of the Laboratory Guide to Molecular Cloning (Third Edition) by [American] J. Sambrook D.W. Russell.
Operation method
In vitro mutagenesis using double-stranded DNA as a template: mutant selection experiment with DpnⅠ
Materials and Instruments
Receptorized E. coli strains with hsdR17 genotype Move makings For more product details, please visit Aladdin Scientific website.
ATP Mixture of four dNTFs Mutagenesis buffer NaOH NaAC TE Phage T4 DNA ligase Phage T4 polynucleotide kinase DpnI Restriction endonuclease Heat-stable DNA polymerase Agarose gel Oligonucleotide primers Plasmid DNA
Automatic micropipette tips with baffles Microcentrifuge tubes Microtitration plates Adjustable pipettes Thermal cyclers with the ability to set the desired workstation expansion parameters
Buffers and solutions
Refer to Appendix 1 for the composition of storage solutions, buffers and reagents.
Dilute the storage solution to the appropriate concentration.
ATP (10 mmol/L)
A mixed solution containing four dNTFs, each with a dNTP concentration of 5 mmol/L
10X Long PCR Buffer (for mixed DNA polymerases)
500 mmol/L Tris-Cl (pH 9.0 at room temperature)
160 mmol/L Sulfuric Acid Press
25 mmol/L MgCl2
1.5 mg/ml BSA
Manufacturer-supplied buffers and heat-stabilized DNA polymerase buffer may be substituted for the above buffers.
OR
10x Mutagenesis Buffer (suitable for Pfu polymerase analogs)
100 mmol/L KCl
100 mmol/L Ammonium Sulfate
200 mmol/L Tris (pH 8.9 at room temperature)
20 mmol/L MgSO4
1% Tirton X-100
1 mg/ml BSA without nucleic acid
NaOH (1 mol/L)/EDTA (1 mmol/L) (optional)
NaAC (3 mol/L, PH 4.8) (optional)
This solution is used as a neutralizing reagent and therefore has a slightly lower pH than the NaAC solution used in normal molecular cloning.
TE (pH 8.0)
Enzyme and buffer
Phage T4DNA Ligase (optional)
Phage T4 polynucleotide kinase (optional)
DpnI restriction endonuclease
Heat-stable DNA polymerase (e.g., PfuDNA polymerase)
The working conditions described in this protocol are suitable for Pfu Turbo DNA polymerase. However, the working conditions can also be adapted to other heat-stable DNA polymerases or polymerase mixtures. Stratagene markets Pfu in three forms: a naturally occurring recombinant enzyme produced by expression of a cloned gene, and PfuTurbo, which consists of a recombinant PfuDNA polymerase and a new heat-stabilizing _factor_ of unknown properties that can improve the yield of amplified products without altering the accuracy of DNA amplification. The PfuTurbo enzyme. The manufacturer claims that Turbo DNA polymerase is capable of amplifying DNA fragments up to 15kb in length. In our experience, the amplification efficiency decreases when the length of double-stranded plasmid DNA exceeds 7-8kb.
Gel
1% agarose gel containing 0.5% ethidium bromide.
See step 8.
Nucleic Acids and Oligonucleotides
Oligonucleotide primers
Advice on oligonucleotide primer design is given in the Introduction to this protocol and in the Information section on Mutagenic Oligonucleotides. Oligonucleotide primers give the best results if purified by FPLC and PAGE to reduce the level of salt contamination (see Scheme 1 or 5 in Chapter 10). Dissolve the purified primers in water at a concentration of 20 mmol/L.
Plasmid DNA
The template DNA used for mutagenesis is a circular plasmid containing the target gene or cDNA. Generally, the smaller the plasmid, the higher the amplification efficiency of the target DNA. Plasmids smaller than 7kb are more efficient; however, plasmids up to 11.5kb in length have also been successfully used as DNA templates. Plasmid DNA was dissolved in 1 mmol/L Tris-Cl with low concentration of EDTA (<0.1 mmol/L) at a concentration of 1ug/ml.
Specialized equipment
Pipette tips for automated micropipettes with baffles
Microcentrifuge tubes (0.5 ml thin-walled tubes for amplification) or microtitre plates
Adjustable pipettes
Thermal cycler capable of setting the desired workstation expansion parameters
If the thermocycler does not have a heated lid, use mineral oil or paraffin oil to prevent evaporation of fluids during PCR.
Other Reagents
The reagents needed for Step 14 of this protocol are listed in Chapter 1, Protocol 23.
The reagents required for step 15 of this protocol are listed in Chapter 1, Option 1.
Reagents needed for step 16 of this protocol are listed in Chapter 12, Options 3, 4, or 5.
Vectors and Bacterial Strains
See Appendix 3.
Receptorized E. coli strains with the hsdR17 genotype (e.g., XLl-Blue, XL2-Blue MRF' or DH5a)
Methods
Amplification of target DNA with mutagenic primers
Steps 1 and 2 are optional (see note after step 3).
1. dissolve 1-10ug of plasmid DNA in 40ul of H20 and add 10ul of 1mol/L NaOH/1 mmol/LEDTA. incubate at 37°C for 15 min to denature the plasmid DNA template.
2. Add 5ul of 3mol/L (pH4.8) to neutralize the above reaction solution, and then add 150ul of pre-cooled ethanol to precipitate the DNA.
3. Collect the denatured plasmid DNA by centrifugation in a microcentrifuge at 4°C for 10 min. Carefully discard the ethanol supernatant and wash the precipitate with 150ul of 70% ethanol. Re-centrifuge for 2 min, discard the supernatant and allow all ethanol to evaporate at room temperature. Resuspend the DNA in 20ul of water.
It is not theoretically necessary to denature the plasmid DNA template. If the base denaturation step is ignored, the superhelical natural double-stranded DNA will be denatured by heating at 94°C during the first cycle of PCR. Steps 1 and 2 are required because of the state of the plasmid DNA after denaturation in an alkaline environment. In 0.2mol/LNaH solution. In 0.2mol/LNaH solution, the plasmid DNA folds into a dense and irreversible denatured state, which makes it very unlikely that the plasmid DNA will be used as a PCR template. The possibility of transforming bacteria is very small. This greatly reduces the number of clones containing unmutated wild-type DNA (Du et al. 1995, Dorrell et al. 1996). In contrast, although the 95°C temperature of the first cycle of PCR breaks the DNA double helix, it does not necessarily destroy the transforming ability of the plasmid DNA. Unmutated parental plasmid DNA molecules were present in a higher percentage of the clones obtained after transformation. The significance of base denaturation did not become apparent until later in the experiment, when researchers were faced with the task of screening clones to identify those containing mutant DNA. If mutagenesis is inefficient and Dpn I screening is ineffective, the proportion of clones containing wild-type DNA molecules is often unacceptably high.
4. Using a sterile 0.5 ml microcentrifuge tube, set up a series of two oligonucleotide primers containing different amounts (e.g., 5, 10, 25, 50 ng) of plasmid DNA and a constant amount of plasmid DNA.
10X Mutagenesis Buffer 5ul
Template Plasmid DNA 5~50ng
Oligonucleotide primer 1 (20 mmol/L) 2.5ul
Oligonucleotide primer 2(20 mmol/L) 2.5ul
dNTP mixture 2.5ul
Add water to 50ul
Add 2.5 units of Pfu Turbo DNA Polymerase.
Note that the reagents are added in the above order to minimize degradation of primers generated by Pfu Turbo 3'-5' exonuclease activity.
5. If the thermal cycler does not have a heated lid, add 1 drop (~50ul) of light mineral oil or 1 drop of paraffin to cover the reaction mixture to prevent evaporation of the sample during repeated heating and cooling cycles. Place the centrifuge tube in the thermal cycler.
6. Perform amplification using the denaturation, annealing, polymerization time and temperature conditions listed in the table. 
NOTE: The above times apply to 0.5 ml thin-walled tubes and reaction systems incubated on a Perkin-Elmer Model 9600 or 9700, Master Cycler (Eppendorf), or PTC100 (MJ Research) thermal cycler only. If other types of equipment and reaction systems are used, the time and temperature required for the reaction will need to be adjusted.
Single base substitution reactions require 12 cycles for linear amplification; 16 cycles for single amino acid substitutions (usually two to three bases in close proximity); and 18 cycles for insertion or deletion of DNA fragments of any size. Insertion or deletion of DNA fragments of any size uses 18 cycles.
P/u catalyzes DNA synthesis 1.5 to 2.0 times slower than Taq in the amplification reaction.
The use of a small number of cycles and a large number of starting templates reduces the number of pseudo-mutations in plasmid DNA, genes, or cDNAs that can be generated during amplification.
7. After the DNA amplification reaction is complete, place the reaction product on ice.
8. Remove 10ul of reaction product from each amplification reaction and electrophoresis the target DNA amplification on a 1% agarose gel (containing O.5ug/ml ethidium bromide). As a rule, 50ng of unamplified linear plasmid DNA and a 1kbDNA gradient molecular weight standard are used as reference.
If the amplification efficiency is low, design a series of reactions to optimize the appropriate cycling parameters and the optimal amount of components required for the amplification reaction.
Ligation and transformation of amplification products
Steps 9 to 12 may or may not be done and are usually only used when mutagenesis efficiency is low (e.g. when constructs are inserted or missing).
9. Amplified DNA is extracted twice with phenol/chloroform and precipitated with ethanol.
10. Resuspend the DNA in:
10X Phage T4 Polynucleotide Kinase Buffer 5ul
10 mmol/L ATP 5ul
Phage T4 Polynucleotide Kinase 5 Units
Add water to 50ul
Incubate at 37°C for 1h, heat at 68°C for 10 min to inactivate the kinase. Phosphorylated DNA was extracted twice with phenol/chloroform and collected by ethanol precipitation.
11. Resuspend the above phosphorylated DNA precipitates (approximately 0.9ug each) in 90ul of TE. Design a series of ligation reactions with phosphorylated DNA concentrations ranging from 0.1 to 1ug/ml.
Phosphorylated DNA (10 to 100ng)
10x Phage T4DNA Ligase Buffer 10ul
10 mmol/LATP 10ul
Phage T4DNA Ligase 4 Units
Add water to 100ul
Incubate at 16°C for 12-16 h. Incubate for 10 h.
If the 10X Phage T4 Ligase supplied by the manufacturer already contains ATP, omit the ATP component of the above ligation reaction.
Although the conditions of the ligation reaction that favor the formation of cyclic monomers have been understood theoretically (Collins and Welssman 1984), it is difficult to do so in practice. The molar concentration of DNA molecules must be very low in order to favor intramolecular cyclization rather than intermolecular linkage. However, when DNA is produced by reverse PCR amplification, it is difficult to calculate the appropriate DNA concentration because it is impossible to know the proportion of full-length DNA with undamaged ends in the PCR product.
12. phenol: The DNA ligations are extracted twice with chloroform and the DNA is collected by ethanol precipitation. the DNA precipitate is resuspended in 45ul of water and 5ul l0x Dpn I buffer is added per tube.
13. Add 10 units of DpnⅠ directly to the remaining amplification reaction from step 7 or to the phosphorylated and ligated DNA from step 12 to digest the DNA. mix the reagents by pipetting up and down several times. Incubate in a microcentrifuge for 5 s at 37 °C for 1 h.
If the PCR reaction is performed in the presence of mineral or paraffin oil, be sure to add DpnⅠ to the aqueous phase of the reaction mixture. Use a shielded micropipette tip, ensuring that the tip of the pipette is inserted under the mineral or paraffin oil cover.
14. Transform the E. coli recipient bacteria with 1, 2, and 5 ul of digested DNA according to the procedure described in Scheme 23 in Chapter 1.
Ensure that no mineral oil is carried from the digestion reaction mixture into the receptor cells. The use of super-receptor E. coli cells XL2-Blue MRF' (Stratagene) and a modified transformation procedure facilitates the shoving process for recovery of mutant transformations (Dorrell et al. 1996). However, in our experience, the laboratory's own E. coli receptor foot can be used for most cyclic mutagenesis experiments.
15. Plasmid DNA is prepared from at least 12 individual transformants; however, if the mutation results in the creation or destruction of a new site or the introduction of an insertion or deletion into the template, the DNA mutants are screened by DNA sequence analysis, oligonucleotide hybridization (Scheme 7), or restriction enzyme digestion of small amounts of plasmid DNA.
16. Sequence analysis of all target DNA fragments to verify that the intended mutant was generated and that no pseudomutants were generated during amplification (see Schemes 3, 4, and 5 in Chapter 12). 