The key features of this PCR-SDM method include the following: 1. the concentration of template used is increased, which reduces the number of cycles and therefore the likelihood of amplification of nonspecific products. 2. the addition of Taq Extender improves the yield and reliability of amplification of longer PCR products. 3. the use of Dpm I restriction endonuclease reduces the number of parent molecules. 4. the use of Pfu DNA polymerase removes the extended bases, improving the efficiency of flat-end joining. The use of Pfu DNA polymerase to remove extended bases improves the efficiency of flat-end joining. This experiment is from the PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.
Operation method
Rapid PCR targeted mutation assay
Principle
The key features of this PCR-SDM method include the following: 1. the concentration of template used is increased, which reduces the number of cycles and therefore the likelihood of amplification of nonspecific products. 2. the addition of Taq Extender improves the yield and reliability of amplification of longer PCR products. 3. the use of Dpm I restriction endonuclease reduces the number of parent molecules. 4. the use of Pfu DNA polymerase removes the extended bases, improving the efficiency of flat-end joining. The use of Pfu DNA polymerase removes extended bases and improves the efficiency of flat-end ligation.
Materials and Instruments
Template DNA Move I. MATERIALS Caveat If mineral oil is used to cover the reactants in the cycle, be sure to insert the tip of the micropipette below the layer of mineral oil when adding additional components to the reaction tubes during digestion, polishing, and ligation. Common Problems Genetic engineering and protein engineering research often uses gene mutation techniques to prepare mutants for the study of gene regulation, expression and protein structure and function. There are two traditional methods used to study the relationship between protein structure and function: (1) modification of amino acid residue side chains in the primary structure of proteins; and (2) X-ray diffraction of protein crystal structures. Although these methods can provide a certain amount of information, they are constrained by many conditions and are of limited use. If the targeted mutagenesis technique is used to introduce mutations at predetermined sites in the cloned DNA, followed by expression of the modified mutant in an appropriate host cell-vector system, structural domains and individual amino acid residues critical to the structure and biological function of the protein can be identified by comparing the properties of the mutant protein with those of the wild-type protein. The development of both targeted mutagenesis and expression of cloned genes has made mutant studies a preferred option for biochemists and molecular biologists to analyze protein structure and function. For more product details, please visit Aladdin Scientific website.
ATP dNTP solution SDM buffer Taq DNA polymerase Cloned Pfu DNA polymerase Taq Extender PCR additive Dpn I restriction endonuclease T4 DNA ligase Template DNA dsDNA plasmid LB agar plate
FALCON 2059 tubing or alternative Thermocycler Water bath or appropriate heating device Pre-set to 37°C, 42°C, 72°C Temperature chamber Agarose gel electrophoresis reagents and devices
1. Buffers, solutions and reagents
ATP (10 mmol/L)
dNTP solution (contains all 4 dNTPs, 6.25 mmol/L each)
SDM buffer, 10X (20 mmol/L Tris-HCl, pH 7.5,8 mmol/L MgCl2, 40ug/ml BSA)
2. Enzymes and enzyme buffers
Taq DNA polymerase
Cloned Pfu DNA polymerase (Stratagene)
Taq Extender PCR additive (Stratagene)
Dpn I restriction endonuclease
T4 DNA ligase
3. Nucleic acid and oligonucleotide mutation primers
Template DNA, dsDNA plasmid, small or large volume preparations [O.5pmol template=(0.33ug /kb) X template size (kb)]
Template DNA must have methylated Gm6 ATC sequences, if not, it must be methylated in vitro with Dam methylase prior to PCR-SDM.
Only plasmids containing bacteriostatic antibiotic resistance genes (e.g., penicillin, tetracycline, and chloramphenicol) are suitable for this rapid protocol, and are also able to be used with plasmids containing bactericidal antibiotic resistance genes (e.g., kanamycin and streptomycin), but the transformed cells must be allowed to have a growth spurt prior to applying antibiotic selection. See note on step 14.
Mutagenesis primers [15pmol primer = (5ng/base) X primer size (bases)]
It is important to have a phosphate group on one or both primers, which are capable of being phosphorylated by the T4 polyribonucleotide kinase or synthesized directly into a 5' terminal phosphate group .
4. Medium
LB agar plates (10 g tyrosine for bacterial culture, 5 g yeast extract for bacterial culture, 10 g NaCl, Add H20 to 1L, final pH 7.0, add 15 g agar per liter)
5. Special equipment
FALCON 2059 tubing or alternative
Thermocycler
Water bath or appropriate heating device, pre-set to 37°C, 42°C, 72°C Warmer, pre-set to 37°C
Small Centrifuge Tubes
6. Vectors and strains
E. coli Heat Shock Susceptor Cells (e.g., XLl-blue, Statagene)
7. Additional items
Options: agarose gel electrophoresis reagents and apparatus, including ethidium bromide (see step 9)
II. METHODS
1. PCR
(1) On ice, in a well sterilized centrifuge tube, mix the PCR-SDM reaction.
Template DNA 0.5pmol
SDM buffer, 10X 2.5ul
dNTP solvent (6.5 mmol/L) 1ul
Mutation primers 15pmol each
H20 Make up to 24ul
(2) Add 2.5U of Taq DNA polymerase and 2.5U of Taq Extender PCR additive.
These enzymes can be mixed together and stored as a 1:1 (v/v) mixture at -20°C for at least 3 months.
(3) Perform 7 to 12 cycles of amplification under the following PCR conditions. 
The time and temperature can be adjusted accordingly for different types of equipment and reaction systems, see 31.2 PCR Precautions.
If the thermocycler does not have a thermal cover, use mineral oil to form a paraffin wax to prevent evaporation of the reaction mixture during PCR.
2. Digestion and polishing of PCR-SDM products
(4) After the PCR reaction, cool the reaction products on ice for 2 min.
(5) Add the following components directly to 25ul of amplification product.
Dpn I Restriction Endonuclease (10U/ul) 1ul
Pfu DNA Polymerase (2.5U/ul) 1ul
If mineral oil was used to cover the reactants during the cycle, be sure to insert the tip of the micropipette below the mineral oil layer when adding additional components to the reaction tube during digestion, polishing, and ligation.
(6) Mix gently and centrifuge the reaction in a centrifuge tube for lmin. Immediately incubate the reaction at 37°C for 30 min.
(7) Incubate the reaction at 72°C for an additional 30 min.
3. Ligation of PCR-SDM products
(8) Add the following components to the product treated with Dpn I and cloned Pfu DNA polymerase.
H2O 100ul
SDM buffer, 10X 10ul
ATP,10mmol/L 5ul
(9) Mix gently and then centrifuge the reaction in a centrifuge tube for 1min.
Optionally, to demonstrate the integrity of the PCR-SDM product, 5ul of the product is removed from the sample and analyzed in a standard agarose gel electrophoresis. A single band should be observed.
(10) 10ul of the above reaction was placed in a sterilized centrifuge tube and 4U of T4 DNA Ligase (4U/ul) was added.
It appears that different batches of T4 DNA ligase have quite different abilities to ligate flat DNA molecules. This leads to variations in mutation efficiency, which can range from 30% to 70% in this method. Once a good quality batch of enzyme is found, it is recommended to save it for exclusive use as PCR-SDM.
(11) Warm the reaction at 37°C for 1h.
4. Transformation of Receptor Cells (Rapid Transformation Protocol)
(12) Gently thaw the heat shocked receptor cells on ice and remove 40ul of the cells into a pre-cooled FALCOL2059 polypropylene tube.
(13) Add 1ul of ligase-treated DNA to the cells, stir gently, and place on ice for 30 min.
(14) Heat-excite at 42°C for 30s, then place on ice for 2 min.
The heat-excitation conditions have already been optimized for the FALCON2059 tubes, and if different tubes are used, the reaction conditions should be re-optimized accordingly.
If the plasmid used contains a bactericidal antibiotic resistance gene, after 2 min on ice, add 260ul of LB and shake at 37°C for 30 min at 250r/min, then continue with step 15.
(15) Immediately place all volumes of sensory cells onto a coated LB agar plate containing the appropriate selective antibiotic. Place the plate at 37°C overnight.
Source "PCR Laboratory Guide (Second Edition)" by Seed Kang and Qu Lijia.