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MOLECULAR BIOLOGY: WORKING WITH DNA

POLYMERASE CHAIN REACTION

HIGH FIDELITY DNA AMPLIFICATION BY PCR USING VENT, PWO AND PFU DNA POLYMERASES

High Fidelity DNA Amplification by PCR using Vent, Pwo, and Pfu DNA Polymerases Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Contributor:	The Laboratory of Jasper Rine at the University of California, Berkeley
Overview Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
This protocol describes the general considerations and experimental conditions for amplification of genes through PCR by using different commercially available DNA polymerases. The author of this protocol established these conditions while searching for methods to improve fidelity and yield of PCR products for use in the yeast two-hybrid screen, and has included a very useful comparison table of the error rates for the different polymerases, including Taq, under the experimental conditions.
Procedure Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
A. General PCR Conditions with Pfu DNA Polymerase 1. Set up the appropriate number of PCR tubes on ice. 2. Add the following components in order:    77 μl of ddH2O    10 μl of 10X Pfu DNA Polymerase Buffer    2 μl of dNTP Mix    3 μl of Oligonucleotide #1    3 μl of Oligonucleotide #2    4 μl of DNA Template    1 μl of Pfu DNA Polymerase (Promega) Add Pfu DNA Polymerase and mix the solution gently by either trituration or by flicking the tube several times. Cover the reactions with a layer of mineral oil if the PCR machine does not have a heated lid. 3. For products less than 2,000 basepairs, use the following temperature cycling profile:    1 cycle of 95°C for 45 sec    15 cycles of 95°C for 45 sec, 45°C for 45 sec, 72°C for 3 min    1 cycle of 72°C for 5 min 4. For products greater than 2,000 basepairs, use the following temperature cycling profile:    1 cycle of 94°C for 2 min    15 cycles of 94°C for 15 sec, 45°C for 30 sec, 72°C for 6 min    1 cycle of 72°C for 5 min 5. Once complete, store reactions at 4°C and analyze products by Agarose gel electrophoresis (see Protocol ID#1265). B. General PCR Conditions with Pwo DNA Polymerase 1. Set up the appropriate number of PCR tubes on ice. 2. Add the following components in order:    75.5 μl of ddH2O    2 μl of dNTP Mix    3 μl of Oligonucleotide #1    3 μl of Oligonucleotide #2    4 μl of DNA Template    2 μl of 25 mM Magnesium Sulfate    10 μl of 10X Pwo DNA Polymerase Buffer with MgSO4 (Boehringer Mannheim)    0.5 μl of Pwo DNA Polymerase Add Pwo DNA Polymerase and mix the solution gently by either trituration or by flicking the tube several times. Cover the reactions with a layer of mineral oil if the PCR machine does not have a heated lid. 3. For products less than 2,000 basepairs, use the following temperature cycling profile:    1 cycle of 94°C for 2 min    15 cycles of 95°C for 15 sec, 43°C for 30 sec, 72°C for 1.5 min    1 cycle of 72°C for 5 min. 4. For products greater than 2,000 basepairs, use the following temperature cycling profile:    1 cycle of 94°C for 2 min    15 cycles of 94°C for 15 sec, 43°C for 30 sec, 72°C for 3 min    1 cycle of 72°C for 5 min. 5. Once complete, store the reactions at 4°C and analyze the products by Agarose gel electrophoresis (see Protocol ID#1265). C. General PCR Conditions with Vent DNA Polymerase 1. Set up the appropriate number of PCR tubes on ice. 2. Add the following components in order:    73.5 μl of ddH2O    2 μl of dNTP Mix    3 μl of Oligonucleotide #1    3 μl of Oligonucleotide #2    4 μl of DNA Template    4 μl of 25 mM Magnesium Sulfate    10 μl of 10X Vent DNA Polymerase Buffer (New England Biolabs)    0.5 μl of Vent DNA Polymerase Add Vent DNA Polymerase and mix the solution gently by either trituration or by flicking the tube several times. Cover the reactions with a layer of mineral oil if the PCR machine does not have a heated lid. 3. For products less than 2,000 basepairs, use the following temperature cycling profile:    1 cycle of 94°C for 2 min    15 cycles of 95°C for 15 sec, 43°C for 30 sec, 72°C for 1.5 min    1 cycle of 72°C for 5 min 4. For products greater than 2,000 basepairs, use the following temperature cycling profile:    1 cycle of 94°C for 2 min    15 cycles of 94°C for 15 sec, 43°C for 30 sec, 72°C for 3 min    1 cycle of 72°C for 5 min 5. Once complete, store the reactions at 4°C and analyze the products by Agarose gel electrophoresis (see Protocol ID#1265). D. Error Rates of Pfu, Pwo, Vent, and Taq DNA Polymerases 1. The above conditions have been used to determine the relative error rates of the different thermo-stable DNA polymerases using a 2,000 base pair DNA template:    Pfu = 1.6 X 10-6    Pwo = 2 X 10-6    Vent = 4 X 10-6    Taq = 2 X 10-5 After 15 cycles, this translates into the following percentages for molecules of product DNA (2 kb) that contain a misincorporated nucleotide:    Pfu = 5%    Pwo = 6%    Vent = 11%    Taq = 46%
Solutions Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
dNTP Mix    10 mM dATP 10 mM dTTP Store at -20°C in small aliquots. 10 mM dCTP 10 mM dGTP Pfu DNA Polymerase Buffer (10X)    1% (v/v) Triton X-100 100 mM KCl 200 mM Tris-Cl, pH 8.8 1 mg/ml Bovine Serum Albumin, nuclease-free 100 mM (NH4)2SO4 20 mM MgSO4 25 mM Magnesium Sulfate    25 mM MgSO4 DNA Template    0.2 mg/ml Plasmid containing DNA of interest Oligonucleotide #2    10 μM 3' oligonucleotide Oligonucleotide #1    10 μM 5' oligonucleotide
BioReagents and Chemicals Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Oligonucleotide DNA Polymerase, Vent DNA Polymerase, Pwo dCTP DNA Polymerase, Pfu dTTP Plasmid dGTP dATP DNA Polymerase, Taq
Protocol Hints Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
1. In general, the polymerases that are the most accurate tend to have the lowest yields of PCR product and require longer extension times. This is because these enzymes possess a 3' to 5' exonuclease activity as part of the mechanism for proofreading. Note that this exonuclease activity is significant when the pool of mononucleotides is low. 2. As with all experiments utilizing PCR, melting temperature, G + C content, and avoidance of duplex formation should be considered when choosing oligonucleotides. Other factors that can be tweaked to optimize product formation are Mg++ concentration, annealing temperature, dNTP concentration, and template concentrations.