The large primer method was originally established by KAMMANN et al. (1989) and the current method has been modified by many researchers including Sarkat and Sommer (1990,1992), Giebel and Spritz (1990), Landt et al. (1990), Marini et al. (1993), Picard et al. Ling and Robinson (1997) and others modified to produce the simplest and least expensive method based on PCR mutagenesis. 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
Efficient and rapid sentinel mutagenesis in the same tube using large-primer PCR
Materials and Instruments
Amplification buffer A mixture of four dNTPs Heat-stabilized DNA polymerase Agarose or polyacrylamide gels Forward and reverse internal primers Mutagenic primers Template DNA Move makings For more product details, please visit Aladdin Scientific website.
Pipette tips for automated pipettes with barrier devices Centrifuge tubes for microcentrifuges Adjustable pipettes
Buffers and solutions
Refer to Appendix 1 for the composition of storage solutions, buffers and reagents.
Dilute the storage solution to the appropriate concentration.
10x Amplification Buffer
Contain a mixture of four dNTPs, each at a concentration of 2.5 mmol/L dNTF
Enzyme and buffer
Heat-stabilized DNA polymerase [Hot-Tub DNA polymerase (Amersham) or equivalent].
Most DNA polymerases are stored in a storage solution containing 50% glycerol. This solution is very viscous and difficult to measure accurately. The simplest method is to centrifuge the enzyme-containing tube in a microcentrifuge at 4°C for 10s at maximum speed and then remove the desired amount of enzyme using an adjustable pipette. PCR reaction components are assembled using an automated pipette with a barrier device.
Gel
1% agarose gel or polyacrylamide gel (containing 0.5ug/ml ethidium bromide).
Nucleic acids and nucleotides
Primers
The forward and reverse internal primers are dissolved in water at a concentration of 100umol/L (100pmole/ul).
Mutagenic primers are dissolved in water at a concentration of 10mmol/L (10pmole/ul).
Template DNA
Purify superhelical double-stranded plasmid DNA by ethidium bromide-cesium chloride density gradient centrifugation (Scheme 10 or 11 in Chapter 1) or by Qiagen resin column chromatography (Scheme 9 in Chapter 1). Dissolve the template DNA in TE (pH 8.0) solution at a concentration of 0.1ug/ml.
Specialized equipment
Pipette tips for automated pipettes with barrier device
Centrifuge tubes for microcentrifuge (0.5 ml thin-walled tubes for amplification)
Adjustable Pipettes
Thermal cycler that can be programmed to the desired amplification conditions
If the thermal cycler is not equipped with a heated lid device, use mineral oil or paraffin oil to prevent volatilization of the reaction mixture during PCR.
Other Reagents
The reagents required for Step 6 of this protocol are listed in Chapter 1, Protocol 17 or 19.
Methods
1. Using a 0.5 ml microcentrifuge tube or amplification reaction tube (on ice), mix the following reagents required for the first round of amplification reactions
Mix the following reagents for the first round of the amplification reaction:
10X Amplification Buffer 10ul
Plasmid DNA template 200~400pg
2.5 mmol/LdNTP solution 8ul
Mutagenic primer 10pmoles
Low Tm side primer lOOpmoles
Heat-stable DNA polymerase 0.5ul (2.5 units)
Add water to 100ul
If the manufacturer's Thermal Stable DNA Polymerase 10x Amplification Buffer does not contain MgCl2, add the desired volume of 0.1mol/L MgCl2 so that the reaction mixture contains the optimal concentration of divalent ions for the DNA polymerase reaction.
2. If the thermal cycler does not have a heated lid, add a drop of paraffin oil (~50ul) to cover the PCR reaction. Place the reaction tube in the thermal cycler.
3. amplify the nucleic acids using the time and temperature required for denaturation, annealing, and polymerization reactions given in the following table 
The above reaction conditions are suitable for 0.5 ml thin-walled tubes and 100ul reaction volumes on a Perkin-Elmer 9600 9700, Master Cycler (Eppendorf) or PTC.100 (MJ Research) thermocycler: the above conditions need to be adapted for use with other types of instruments or different reaction volumes. The polymerization reaction should be carried out for 1 min per 1000 bp length of target DNA.
4. After the first PCR reaction, add the following components to the same reaction tube:
Side primers with high Tm values 100pmoles
Heat-stabilized DNA polymerase 0.5ul (2.5 units)
2.5 mmol/LdNTP solution 3ul
Mix the above reaction with a few gentle up and down strokes with a pipette. It can be centrifuged for a few seconds to collect all the reagents to the bottom of the centrifuge tube.
3-5ul of the first PCR reaction mixture can be taken and quickly analyzed by agarose gel electrophoresis to confirm the successful synthesis of the macroprimed PCR product.
5. A second amplification reaction consisting of 25 cycles and a two-step temperature is performed:
94°C, 40S
72°C,90s The final extension step is 72°C,5 min.
6. 5% of the second PCR amplification reaction is subjected to agarose gel electrophoresis or polyacrylamide gel electrophoresis to estimate the concentration of target DNA amplified.
If restriction enzyme sites are set in the primers, digest the amplified DNA with the appropriate restriction enzyme, and then subclone into a suitable vector. Alternatively, the amplified DNA phosphorylated in step 5 can be ligated into a plasmid vector that has been digested with restriction enzymes to produce pure ends. This method has a mutation rate of about 80%, and usually only 6 clones need to be selected for DNA sequence analysis to confirm the presence of the desired mutant, and to make sure that there are no other mutations in the full DNA sequence.