Overlapping extensions generate site-specific mutagenesis experiments
Overlapping extensions generate site-specific mutagenesis experiments
Four primers are required for site-specific mutagenesis by the overlapping extension method (see Fig. 13-4) (Higuchi et al. 1988, Hetal. 1989). 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
Overlapping extensions generate site-specific mutagenesis experiments
Materials and Instruments
Amplification buffer Heat-stabilized DNA polymerase Agarose or polyacrylamide gels Oligonucleotide primers Template DNA Move makings For more product details, please visit Aladdin Scientific website.
Automated pipette tips with barrier device Microcentrifuge tubes Enzymes Adjustable pipettes Thermal cyclers that can be programmed to the desired amplification conditions
Buffers and solutions
For storage solutions, buffer components and reagents required, please see Appendix 1.
Dilute and release the stock solution to the appropriate concentration.
10x Amplification Buffer
Contains a mixture of four dNTPs at 20 mmol/L each.
Enzyme and Buffer
Heat-stabilized DNA polymerase
To avoid misincorporation of bases, a highly efficient heat-stabilized DNA polymerase with 3'-5' exonuclease proofreading capability should be used in the overlap extension mutagenesis reaction. In addition, the DNA polymerase used must not be heat-stabilized. The DNA polymerase used must not have the ability to catalyze non-template incorporation of adenylate residues. DNA polymerases with this property include Puo DNA polymerase (Boehringer Mannheim), rTth DNA polymerase XL (Pwkin Elmer), VentR DNA polymerase (New England Biolabs) and Pfu DNA polymerase (Stmagene).
Heat-stable DNA polymerase mixtures designed for long fragment PCR reactions are also suitable for overlap extension mutagenesis.
Gels
Agarose gel (1%) or polyacrylamide gel containing 0.5ug/ml ethidium bromide.
See steps 6 and 11.
Nucleic Acids and Nucleotides
Oligonucleotide Primers
Each primer should be 20-30 nucleotides in length. They should contain approximately the same number of all four bases, with an even distribution of G and C residues and a low tendency to form stable secondary structures. There should be at least 15 bases of overlapping sequence between the mutagenic primers FM and RM (see Figure 13-4), and the mismatched bases should be located in the middle of the primer sequence. The primers at the 3' and 5' ends of the amplified DNA fragments (i.e. primers with wild-type sequences R2 and F2) can be set with a single restriction site to facilitate cloning of the mutagenized DNA fragments in subsequent steps. For general principles of primer design, see the Introduction section in Chapter 10 on nucleotide primer design.
Oligonucleotide primers synthesized on a DNA autosynthesizer generally do not require purification and can be used directly in overlap mutagenesis.
Template DNA
The template used in mutagenesis is usually plasmid DNA containing the gene or cDNA of interest. the DNA is dissolved at a concentration of 1ug/ml in a 10mmol/L Tris-Cl (pH7.6) solution containing a low concentration of EDTA (<0.1 mmol/L).
Specialized equipment
Automatic pipette tips with barrier device
Microcentrifuge tubes (0.5 ml thin-walled tubes for amplification)
Adjustable pipettes
Thermal cycler that can be programmed with 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 liquid during PCR.
Other Reagents
The reagents required for Step 11 of this protocol are listed in Chapter 1, Protocol 17 or 19.
The reagents required for step 12 of this protocol are listed in Chapter 12, Scheme 3, 4 or 5.
METHODS
1. Oligonucleotide primers FM, RM, R2 and F2 are designed and synthesized according to the requirements in Materials and Methods and known DNA sequences.
2. Mix the following reagents in a 0.5 ml microcentrifuge tube or amplification tube to form PCR Reaction 1.
Approximately 100ng of template DNA
10x Amplification Buffer 10ul
20 mmol/L 4 dNTP mix 1.0ul
5umol/L Primer FM(30pmoles) 6.0ul
5umol/L primer R2(30pmoles) 6.0ul
Heat stabilized DMA polymerase 1~2 units
Add water to 100ul
3. Mix the following reagents in a second microcentrifuge tube or amplification tube to form PCR Reaction 2.
Approximately 100ng of template DNA
10x Amplification Buffer 10ul
20 mmol/L 4 dNTP mix 1.0ul
5umol/L Primer RM(30pmoles) 6.0ul
5umol/L primer F2(30pmoles) 6.0ul
Heat-stabilized DNA polymerase 1~2 units
Add water to 100ul
4. If the thermal cycler does not have a heated lid, cover the PCR reaction solution with a drop of paraffin oil (about 50ul). Place the reaction tube in the thermal cycler.
5. Amplify the DNA fragments using the times and temperatures for denaturation, annealing and polymerization given in the table below. 
The above reaction conditions are suitable for 0.5 ml thin-walled tubes and 100ul reaction volumes, as well as the Perkin-Elmer 9600, 9700, Master Cycler (Eppendorf) or PTC 100 (MJ Research) thermal cycler. Adjustment of the above reaction conditions is required when using other types of instruments or different reaction volumes.
The time required for the polymerization reaction should be obtained by calculating the polymerization efficiency of the heat-stabilized DNA polymerase and the length of the DNA template.
The temperature of the annealing reaction should be adjusted according to the mutagenic primer sequence.
6. 5% of the product of each of the above two PCR reactions should be analyzed by agarose or polyacrylamide gel electrophoresis to estimate the concentration of the amplified target DNA.
7. The PCR products are purified using one of the methods described in Chapter 5. Purification of the product tends to increase the yield of the desired amplification product in step 8 and reduces the amount of incorrectly amplified product. It also reduces the background of incorrectly amplified products. (Optional)
8. Mix the following reagents in a sterilized 0.5 ml microcentrifuge tube or amplification tube for the amplification reaction, ligating the 5' and 3' ends of the target gene.
PCR1 amplification product (step 2) approx. 50ng
PCR2 amplification product (step 3) approx. 50ng
10x Amplification Buffer 10ul
5umol/L Primer F2 (know pmoles) 6.0ul
5umol/L primer R2(30pmoles) 6.0ul
Heat stabilized DNA polymerase 1~2 units
Add water to 100ul
9. If the thermal cycler does not have a heated lid, cover the PCR reaction solution with a drop of light paraffin oil (about 50ul). Place the reaction tube in the thermal cycler.
10. Perform amplification using the times and temperatures given in step 5 for denaturation, annealing, and polymerization reactions.
11. Take 5% of the PCR reaction product and perform agarose or polyacrylamide gel electrophoresis to estimate the concentration of amplified target DNA.
If restriction sites are included in the primer F2 and R2 sequences, digest the amplified DNA with these restriction enzymes and subclone them into the appropriate plant. Alternatively, the DNA fragments amplified in step 10 can be phosphorylated and ligated into a vector that has been cut to blunt ends with restriction enzymes.
12. Confirm the full sequence of the amplified DNA fragments after cloning to ensure that no mutations other than those carried by primers FM and RM have been generated during the procedure.
All DNA polymerases show a measurable rate of misincorporation in the in vitro reaction.