pcr-polymerases | PCR Station

PCR ENZYMES: PCR Polymerases

PCR Polymerases - The Ultimate Guide

PCR Polymerases: An Introduction

The choice of the DNA polymerase employed by PCR is determined by the goals of the experiment. There are now a plethora of commercially available enzymes to choose from that differ in their thermal stability, processivity, and fidelity. The most commonly used and most extensively studied enzyme is Taq DNA polymerase.

Initial PCR Polymerases

The DNA polymerases used originally in the first PCR reactions were extracted from the bacterium Escherichia coli. Although this enzyme was an invaluable tool for many molecular biology research uses in the past and even today, it had many disadvantages in the original PCR. In PCR, the pcr reaction must be denatured by heating the double-stranded DNA product after each cycle. Unfortunately, heating also irreversibly inactivated the E. coli DNA polymerase. Fresh aliquots of enzyme had to be added manually at the start of each cycle. With 30-40 cycles of PCR, this was a very laborious and boring task.

What was required was a DNA polymerase that remained stable during the DNA denaturation step performed at 90°C or hotter. A discovery made the solution much easier. The bacterium Thermophilus aquaticus was isolated from water hot springs. This bacterium was able survive and proliferate in the extremely high water temperatures of the hot springs. Isolation of the DNA polymerase from this bacterium yielded a PCR polymerase that was not rapidly inactivated at high temperatures. Gelfand et al. at Cetus corporation successfully purified and cloned this PCR polymerase now called Taq (Thermophilus aquaticus in short) Polymerase.

This allowed a 30-40 cycles of PCR amplification to be performed without the need to open the PCR reaction tube and add fresh polymerase. This also reduced potential contamination that could be introduced when adding polymerase manually over 20-30 times in the original PCR reactions. Also, due to the nature of the thermophilic bacterium and the polymerase, Taq functioned optimally at temperatures around 72°C, allowing DNA synthesis to be performed at much higher

temperatures than was possible with the E. coli enzyme. This had the advantage of allowing the template DNA strand to be copied at a much higher fidelity due to higher strigency of PCR primer annealing. This further reduced non-specific products which had affected many earlier PCR reactions.

TAQ DNA Polymerase

Taq Polymerase also simply termed "Taq", is a thermostable DNA polymerase used in polymerase chain reaction (PCR) to catalyze the DNA replication reaction in the PCR cycle. In this manner of amplification, PCR is able to examine for the presence or absence of a gene of interest in a biological sample.

Taq DNA polymerase

Molecular model of Taq DNA Polymerase. Crystal structure of Taq DNA-Polymerase Shows A New Orientation For The Structure-Specific Nuclease Domain.

Taq DNA polymerase replaced E.Coli DNA Polymerase in PCR, due to its thermostable properties. Taq was first isolated from Thermus aquaticus, a bacterium that lives in hot springs and hydrothermal vents.

Taq was the first polymerase that was able to withstand the denaturing conditions (over 90 °C) required during PCR cycling.

Taq has an e nzymatic halflife at 95°C of about 40 min. For more properties of Taq, see the table below.

One of Taq polymerases' major disadvantages is its low replication fidelity. As Taq does not have 3' to 5' exonuclease proofreading mechanism to replace an accidental mismatch in the newly synthesized DNA strand, Taq produces more errors than proofreading polymerases such as Pfu.

Taq DNA polymerase also is unique in that it produces PCR products with A (Adenine) overhangs. This was found to be quite useful, and was exploited to produce TA Cloning and TOPO cloning (Invitrogen). These methods employ a cloning vector (or plasmid) which possesses T (Thymine) overhangs. This allows ligation using topoisomerase or DNA ligase to quickly be accomplished with the A overhangs of the PCR product

PFU DNA Polymerase

Pfu DNA polymerase is an enzyme found in the hyperthermophilic archaeon Pyrococcus furiosus, where it functions in vivo to replicate the organism's DNA. In vitro, Pfu is used to quickly amplify DNA in the Polymerase Chain Reaction, where the enzyme serves the central function of copying a new strand of DNA during each extension step.

Superiority of Pfu polymerase The main difference between Pfu and alternative enzymes is Pfu's superior thermostability and 'proofreading' properties compared to other thermostable polymerases. Unlike Taq DNA polymerase, Pfu DNA polymerase possesses 3' to 5' exonuclease proofreading activity, meaning that it works its way along the DNA from the 3' end to the 5' end and corrects nucleotide-misincorporation errors. This means that Pfu DNA polymerase-generated PCR fragments will have fewer errors than Taq-generated PCR inserts. As a result, Pfu is more commonly used for molecular cloning of PCR fragments than the historically popular Taq. Commercially available Pfu typically results in an error rate of 1 in 1.3 million base pairs and can yield 2.6% mutated products when amplifying 1kb fragments using PCR. However, Pfu is slower and typically requires 1–2 minutes to amplify 1kb of DNA at 72° C. Using Pfu DNA polymerase in PCR reactions also results in blunt-ended PCR products. Pfu DNA polymerase is hence superior for techniques that require high-fidelity DNA synthesis, but can also be used in conjunction with Taq polymerase to obtain the fidelity of Pfu with the speed of Taq polymerase activity.

History

Scientists associated with the biotech company Stratagene, based in La Jolla, California discovered the superiority of Pfu over Taq in 1991. They published their work in the journal Gene in December of that year (Gene. 1991 Dec 12;108(1):1-6). U.S. Patent 5,489,523 was granted over exonuclease-deficient Pfu in February 1996 while U.S. Patent 5,545,552 over Pfu itself was granted in August 1996.

PCR Polymerases

Summary of Available PCR Polymerases and their properties.

DNA Polymerase

Biological Source

5'--3' Exonuclease

Activity

3'--5' Exonuclease

Activity

95°C Half-life (min)

Commercial Names

Supplying Companies

Extension Rate (nucleosides/s)

Error Rate

Time (s) to 1kb (72°C)

Taq

Thermus Aquaticus

AmpliTaq, AmpliTaq Gold

Promega, Roche, Invitrogen and many others.

1 in 10^3

Pfu

Pyrococcus furiosus

120

PfuTurbo

Stratagene, Fermentas, Invitrogen among others

1 in 1.3 x 10^6

60-120

Pwo

Pyrococcus woesei

N/A

Tfl

Thermas flavus

rTth

Themus thermophilus

Tli

Thermus litoris

400

Vent

Tma

Thermotoga maritima

>50

PCR and Polymerases

PCR has been performed on DNA larger than 10 kilobases, however the average PCR is only several hundred to a few thousand bases of DNA. The problem with long PCR is that there is a balance between accuracy and processivity of the enzyme. Usually, the longer the fragment, the greater the probability of errors.