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 μl.