Long and accurate polymerase chain reaction
In 1992, investigators working on mixing and fusing various domains of different DNA polymerases showed that, when combined with the robust reliability of Taq DNA polymerase, these enzymes produced longer amplicons. Long and accurate polymerase chain reaction (LA PCR) refers to the production of amplified product longer than 3 kilobases (kb) with high fidelity. Long PCR mixtures typically yield PCR products with some tenfold fewer mutations1 than those observed in products resulting from conventional PCR. The mixture of DNA polymerases typically included either Taq or Klentaq1 (which have no 3'-exonuclease proofreading activity) as the major component and, as the minor component, an archaebacterial DNA polymerase (with proofreading activity) such as Deep Vent, Vent or Pfu1. Among other factors that improve LA PCR are the enzyme deoxyuridine triphosphatase (dUTPase)2, which prevents the incorporation of dUTP, the deaminated form of deoxycytosine triphosphate (dCTP) into DNA, and the chemical betaine. Although introduced for amplification of high G-C content targets3, betaine, when included at surprisingly high concentrations, usually helps to promote long PCR up to at least 20 kb. Since the introduction of mixtures of DNA polymerases, most PCRs of any length have improved in reliability and in yield of product. The following protocol is based on the use of ten various primer pairs for the amplification of a genomic template DNA of  5 kb in a reaction volume of 50  l.
Assembly of the reaction
1| Combine the following components of the master mix on ice and mix thoroughly:
10 KLA reaction buffer (pH 9.2): 500 mM Tris base, 160 mM (NH4)2SO4, 25 mM MgCl2, 1% Tween 20. Note, the pH is 9.2 (the optimal for long PCR) without adjustment; some vectors may require pH 7.2. 10-40 dNTP mix: 10 mM each dNTP, 40 mM MgCl2; dissolve the dry form of each dNTP in water to 100 mM stock solutions and store at -80 °C.
2| Remove 48  l to serve as the no-template control reaction and add 2 l of primer pair 1.
3| Add 5.5 l of genomic DNA at 10 ng/l to the master mix (Step 1) and mix thoroughly.
4| Distribute 48 l to each of ten reaction tubes.
5| To each of the ten reaction tubes, add 1 l of each primer (or 2 l of each primer pair), at 10 pmol/l to the corresponding reaction.
There is no need to mix, as this will occur with convection during the cycling.
Amplification
6| Program the thermal cycler as follows. To minimize melt time, ensure that the cycler remains at temperature for no more than 5–10 s for the heat step of each cycle.
The initial melting temperature of 93 °C should be used only for genomic DNA. For other templates, the use of lower temperatures (80 °C) is advised to avoid depurination. The precise annealing temperature is determined by the melting temperature of the primer.
7| Carry out amplification, using the following table to estimate reasonable cycle numbers, depending on the type of template used.
Source
This protocol was adapted from "Long and Accurate PCR" in PCR Primer: A Laboratory Manual (eds. Dieffenback, C.W. & Dveksler, G.S.) 53–60 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2003; http://www.cshlpress.com/link/pcrprm2p.htm).
REFERENCES
- Barnes, W.M. PCR amplification of up to 35 kb DNA with high fidelity and high yield from
 bacteriophage templates. Proc. Natl. Acad. Sci. USA 91, 2216–2220 (1994). | PubMed | ChemPort |
- Lasken, R.S., Schuster, D.M. & Rashtchian A. Archaebacterial DNA polymerases tightly bind uracil-containing DNA. J. Biol. Chem. 271, 17692–17696 (1996). | Article | PubMed | ISI | ChemPort |
- Rees, W.A., Yager, T.D., Korte, J. & von Hippel, P.H. Betaine can eliminate the base pair composition dependence of DNA melting. Biochemistry 32, 137–144 (1993). | Article | PubMed | ISI | ChemPort |
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