The PCR parameters were: Initial denaturation: 94☌ for 5 min, 35 cycles of denaturation: 94☌ for 40 seconds, annealing: 68☌ for 40 seconds, extension: 72☌ for 40 seconds, and final extension: 72☌ for 5 minutes. Different titration stocks of the Pfu Polymerase were made accordingly by dilution with the Pfu buffer (2×, 5×, 10×, 20×).

To estimate the enzymatic activity of the Pfu Polymerase, the band intensity of the PCR amplification was compared to that of the commercial Pfu Polymerases from Promega (3U/μl) (Promega, WI, USA) and Thermo Scientific Fisher (2.5U/μl) (Thermo Scientific, MA, USA). The PCR reactions carried out were in 20 μl volume containing 20 mM Tris-Cl pH 8.8, 10 mM KCl, 10 mM (NH 4) 2 SO 4, 2 mM MgSO 4, 0.1% Triton® X-100 and 0.1 mg/ml nuclease-free BSA, 0.2 mM dNTPs, 0.6 μM p600 primers, and 150 ng of the template. To assess the functionality of the purified Pfu Polymerase, PCR amplification was carried out using the template p600 plasmid (recombinant plasmid containing a 600 bp insert of ICR mouse c-myc gene). The work aims to obtain purified Pfu DNA polymerase at a low cost using a simple in-house purification method that averts specialized techniques and equipment for research and teaching use.įunctional assay of the Pfu Polymerase based on PCR amplification. As a test of functionality, the protein was used to amplify a template plasmid. The overexpressed Pfu DNA polymerase containing lysate was subjected to heating which denatures the rest of the less thermally stable protein, forming debris which is subsequently removed. coli BL21 (DE3) pLysS which capitalized on the thermal stability of the Pfu DNA polymerase as the key strategy for the purification which averts the usage of other tedious, specialized and expensive strategies. In this study, we reported an in-house expression and purification method of Pyrococcus furiosus (Pfu) DNA polymerase in E. Various researchers have highlight on the expression and purification strategy of the polymerase with a focus on the biophysical characteristic of the protein and/or recombinant DNA technology such as the immobilized metal affinity-tagged purification, weak cation exchange resins, polyethylenemine precipitation, ammonium sulfate precipitation and ion exchange chromatography ( 6– 11). The polymerase possesses a 3′-5′ exonuclease activity that allows for removal of mis-incorporated bases (“proof-reading”) and has been reported to exhibit a low error rate in PCR (1.3×10 −6 mutations/bp/duplication) which is approximately 8-fold lower as compared to Taq Polymerase ( 2, 5). The Pfu DNA polymerase gene encodes a polypeptide of 775 amino acids with a predicted molecular weight of 90, 109 Da ( 3, 4). In applications that require high fidelity such as cloning, a polymerase capable of proof-reading is highly desirable and one such DNA polymerase is the DNA Polymerase from Pyrococcus furiosus ( Pfu). This polymerase has no proof-reading capacity (3′-5′ exonuclease), with an error rate of 8×10 −6 mutations/bp/duplication ( 2). Commonly, Taq polymerase is used as the enzyme of choice in the PCR amplification. Generic PCR consists of three main thermal steps: 1) DNA template denaturation where the double stranded DNA is denatured into two single stranded DNA 2) primer annealing where the primers anneal to the complementary sequences in the template strands 3) and elongation - DNA polymerase synthesizes new copies of DNA from the template ( 1). DNA amplification using Polymerase Chain Reaction (PCR) has long emerged as one of the fundamental technologies of molecular biology.

Pfu ultra touchdown pcr