Pfu DNA Polymerase

exhibits the lowest error rate of any thermostable DNA polymerase studied Detailed Pdsroduct Description

Synonym	Pfu DNA Polymerase
Molecular Formula	N/A
Molecular Weight	N/A
CAS Number	N/A

Specification

Pfu DNA Polymerase exhibits the lowest error rate of any thermostable DNA polymerase studied (1-3). For routine PCR, where simple

detection of an amplification product or estimation of the product's size is important, Taq DNA polymerase is the obvious enzyme to

choose. However, when the amplified product is to be cloned, expressed or used in mutagenesis studies, Pfu DNA Polymerase is a much

better enzyme of choice for PCR. Pfu DNA Polymerase is also used in blends with Taq DNA polymerase, or amino-terminally truncated

versions of Taq DNA polymerase, to amplify longer stretches of DNA in PCR with greater accuracy than Taq DNA polymerase alone

(1).

INTRODUCTION

Pyrococcus furiosus Vc1 (DSM3638) was discovered in geothermally heated marine sediments in Vulcano, Italy (4), and grows

optimally at 100°C. The DNA polymerase was isolated from P. furiosus and shown to possess a 3´-->5´ proofreading exonuclease (5). It

was also shown that the Pfu DNA Polymerase(a) could be used in PCR and that it possessed dramatically better fidelity than Taq DNA

polymerase. The gene encoding the Pfu DNA polymerase was cloned, sequenced and shown to be homologous to the alpha-like DNA

polymerase family (6), and not the Pol I-like family of which Taq DNA polymerase is a member. The Pfu DNA polymerase gene

encodes a polypeptide of 775 amino acids with a predicted molecular weight of 90,109Da (7). We have purified the enzyme from the

native organism and verified its fidelity and performance in PCR. Figure 1 shows the purity and relative sizes of Promega's and another

supplier's enzyme.

Figure 1. Comparison of Promega's Pfu DNA Polymerase protein to that of another supplier by SDS-PAGE. The greater purity of Promega's

Pfu DNA Polymerase is evident by the prominent protein band of ~90kDa in lane 1. Equal activity units of each enzyme preparation were resolved

on a Tris-Glycine 4-20% NovexTM SDS-PAGE gel (220V for 60 minutes in standard Laemmli buffer). Lane 1, Promega's Pfu DNA Polymerase

(native); lanes 2 and 3, separate lots of another supplier's native Pfu DNA polymerase. Lane M, NovexTM Mark 12TM Marker (10μl).

FIDELITY

The low error rate of Pfu DNA Polymerase in PCR, documented and confirmed by several different methods (Table 1), is roughly 106

per base pair (bp) duplicated. Error rate is commonly expressed as the mutation rate per bp duplicated, and accuracy as the inverse of

error rate. In other words, accuracy is the average number of nucleotides the polymerase incorporates before making an error. One of the

higher reported fidelity values for Taq DNA polymerase is 8 x 106 (1). In that study, the fidelity of many thermostable DNA

polymerases was measured using the same method, which demonstrated the superior accuracy of Pfu DNA polymerase for PCR

applications requiring high fidelity (1; Table 2).

rate per bp

Accuracy x

105 (Error rateWe

compared the fidelity of native Pfu DNA Polymerase from Promega with that of another supplier using a PCR-based forward

mutation assay of the E. coli lacI gene (Figure 2). The method is similar to that used by Cline et al. (1). Each PCR was prepared using

the supplied reaction buffer and according to the respective manufacturer's recommendations. Reactions were performed in duplicate as

described in Table 3. We found the accuracy of Promega's native Pfu DNA Polymerase to be 15 times greater than that of the other

supplier's native Pfu DNA polymerase. The increased accuracy of Promega's Pfu DNA Polymerase can be attributed to the difference in

the pH of the reaction buffers provided by each supplier, and is consistent with the data demonstrating the influence of pH on Pfu DNA

polymerase accuracy presented in Cline et al. (1).

Pfu DNA Polymerase