Biotech Glossary | Bioinformatics | Lab Protocol | Notes | Malaysia University |
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Long-Range Polymerase Chain Reaction Reagents and Guidelines |
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| Contributor: |
The Laboratory of George Church at Harvard University | ||||||||||||||||||
| Overview |
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| Efficient Long-Range Polymerase Chain Reaction (PCR) results from the use of two polymerases. A non-proofreading polymerase is the main polymerase in the reaction, and a proofreading polymerase containing 3' to 5' exonuclease activity is present at a lower concentration. This protocol uses the Tth Polymerase (ABI/Perkin-Elmer) as the main-component polymerase and Vent Polymerase (New England Biolabs) as the fractional-component polymerase. Other combinations of proofreading and non-proofreading polymerases have been used successfully. The Long PCR Buffer listed below works well with Tth Polymerase and Vent Polymerase, but may not work for other polymerase combinations. | |||||||||||||||||||
| Procedure |
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A. General Guidelines for Long-Range PCR Conditions and Enzyme Mixtures For Long-Range PCR with low-complexity templates (e.g., plasmid and cosmid inserts): 10 μl of 5X Long PCR Buffer 2 to 5 Units of Tth Polymerase 0.02 Units of Vent Polymerase 5 μl of d10X dNTP Mix (final concentration, 0.2 mM) 5 μl of 10X Magnesium Acetate (final concentration, 1.1 to 1.3 mM) 20 to 40 pmoles each oligonucleotide primer (final concentration, 400 to800 nM) 1 X 105 to 107 template DNA molecules ddH2O to a final volume of 50 μl For Long-Range PCR with moderate-complexity templates (e.g., bacterial genomic DNA): 10 μl of 5X Long PCR Buffer 2 to 5 Units of Tth Polymerase 0.1 Units of Vent Polymerase 5 μl of d10X dNTP Mix (final concentration, 0.2 mM) 5 μl of 10X Magnesium Acetate (final concentration, 1.1 to 1.3 mM) 20 to 40 pmoles each oligonucleotide primer (final concentration, 400 to800 nM) 105 to 107 template DNA molecules ddH2O to a final volume of 50 μl Also see Hint #1 Tricine buffer works well with Tth Polymerase but not as well with Taq Polymerase. The pH of the Tricine Buffer is critical for the efficiency of amplification of long targets. B. Cycle Times and Temperature Considerations Use two temperature cycles with an annealing and extension step at 68°C and a short melting step at 94°C. A rough formula for calculating the annealing and extension time is N = 1 min + (2.5 sec per 100 bases). A constant time of one minute is probably necessary for primer annealing and extension to occur. At 68°C the kinetics of primer-template annealing and melting may become the limiting factor in the rate of primer extension. C. Generic Long-Range PCR Program Initial denaturing: 94°C for 10 to15 sec Cycles 1 to15: 94°C for 10 sec 68°C for N min Cycles 16 to 30: 94° for 10 sec 68°C for N min: for each cycle add 15 sec to the time, see Hint #2 D. Hot Starts for Long-Range PCR The contributor of this protocol always uses a "hot start" for this Long-Range PCR protocol 1. Split the reaction into two parts: a template/primer fraction, which is 3/4 or 4/5 of the total reaction volume (37.5 to 40 μl), and a DNA Polymerase fraction, which constitutes the remaining 1/4 or 1/5 of the reaction (12.5 to 10 μl). 2. Each fraction should contain 1X Buffer concentration. The DNA Polymerase fraction contains only DNA Polymerase, 1X Buffer, and ddH2O. All other components should be in the template/primer fraction. 3. Incubate the template/primer fraction in the PCR machine at 94°C for 10 sec to denature. 4. Add the DNA Polymerase fraction during the first Annealing - Extension (Cycle 1). 5. Alternatively, after the initial denaturing, an 80°C step can be used for adding the DNA Polymerase fraction. F. Oligonucleotide Primer Selection Use the following general guidelines for designing successful oligonucleotide primersUse primers that are 20 to 23 bases in length Total C and G content should be 12 bases Total A and T content should be 8 to 11 bases The ideal melting temperature (Tm) is between 60 to 68 °C in an 85 mM salt solution (see Hint #3) Avoid primer hairpins Avoid primers with 3' complementarity since this can result in the formation of primer-dimers. Hairpins and primer-dimers can be avoided with the aid of a primer-picking program like PrimerSelect or Oligo. |
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| Solutions |
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| BioReagents and Chemicals |
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Potassium Acetate Oligonucleotide Primer Glycerol DNA Polymerase, Tth DMSO Tricine DNA Polymerase, Vent Magnesium Acetate dCTP dTTP dGTP dATP |
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| Protocol Hints |
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1. The Vent Polymerase concentration of 0.1 Units per reaction has not been completely optimized by the contributing author. A higher concentration of Vent Polymerase may improve the results. According to the contributor of this protocol, other investigators have also found that this concentration of Vent Polymerase works well for Long-Range PCR with high-complexity templates (e.g., human genomic DNA). 2. The 15 sec cycle extensions for cycles 16 to 30 may be necessary for only the longest PCR products (i.e., greater than 20 kb). The contributor suggests determining the cycle extension time empirically. As an example of the 15 sec extension time, assume that the calculated N is 2 min: Cycle 16: 2 min Cycle 17: 2 min 15 sec Cycle 18: 2 min 30 sec etc. 3. A primer can have a Tm significantly lower than 60°C but generally Long-Range PCR will be most effective at Tm above 60°C. The contributor of this protocol has had successful results using primers with Tmvalues below 50°C. The positive control primers from the PCR-XL kit (Perkin-Elmer) and PrimerSelect (DNAStar), both with Tm values of approximately 60°C, have worked successfully in this method. |
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| Citation and/or Web Resources |
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1. Cheng, S., Fockler, C., Barnes, W., Higuchi, R. Effective amplification of long targets from cloned inserts and human genomic DNA. (1994) Proc. Natl. Acad. Sci. 91, 5695-5699. |
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