Long-Range Polymerase Chain Reaction

Conventional polymerase chain reaction (PCR) enables reliable amplification of 3–4 kb of DNA (1) while attempts at optimization has enabled 15.6 kb of λ DNA to be amplified (2). The maximum amplifiable length of PCR is limited by the low fidelity of the Thermus aquaticus (Taq) DNA polymerase (3), the most commonly used thermostable polymerase. It is believed that inadvertent nucleotide misincorporations during the PCR extension steps cause chain terminations (3). The Taq polymerase lacks proofreading properties (4) and thus is unable to correct such misincorporations. The higher extension KM value for a misincorporated nucleotide is thought to cause detachment of the Taq polymerase from template DNA.