Protein model for Taq polymerase (PDB)

T. aquaticus is a bacterium that lives in hot springs and hydrothermal vents, and Taq polymerase was identified^[1] as an enzyme able to withstand the protein-denaturing conditions (high temperature) required during PCR^[2]. Therefore it replaced the DNA polymerase from E.coli originally used in PCR ^[3]. Taq's temperature optimum for activity is 75-80°C, with a halflife of 9 minutes at 97.5°C, and can replicate a 1000 base pair strand of DNA in less than 10 seconds at 72°C^[4].

One of Taq's drawbacks is its relatively low replication fidelity. It lacks a 3' to 5' exonuclease proofreading activity^[4], and has an error rate measured at about 1 in 9,000 nucleotides^[5]. Some thermostable DNA polymerases have been isolated from other thermophilic bacteria and archaea, such as Pfu DNA polymerase, possessing a proofreading activity, and are being used instead of (or in combination with) Taq for high-fidelity amplification.

Taq makes DNA products that have A (Adenine) overhangs at their 3' ends. This may be useful in TA Cloning, whereby a cloning vector (such as a plasmid) is used which has a T (Thymine) 3' overhang, which complements with the A overhang of the PCR product, thus enabling ligation of the PCR product into the plasmid vector.

Taq polymerase in PCR

In the early 1980s Kary Mullis was working at Cetus Corporation on the application of synthetic DNAs to biotechnology. He was familiar with the use of DNA oligonucleotides as probes for binding to target DNA strands, as well as their use as primers for DNA sequencing and cDNA synthesis. In 1983, he began using two primers, one to hybridize to each strand of a target DNA, and adding DNA polymerase to the reaction. This led to exponential DNA replication ^[6], greatly amplifying the amounts of DNA between the primers^[3].

However, after each round of replication the mixture needs to be heated above 90ºC to denature the newly formed DNA, allowing the strands to separate and act as templates in the next round of amplification. Unfortunately, this heating step also inactivates the DNA polymerase then being used, the Klenow fragment of the DNA Polymerase I from E. coli.

Use of the thermostable Taq polymerase eliminates the need for having to add new enzyme to the PCR reaction during the thermocycling process. A single closed tube in a relatively simple machine can be used to carry out the entire process. Thus, the use of Taq polymerase was the key idea that made PCR applicable to a large variety of molecular biology problems concerning DNA analysis^[2].

Significance

Hoffmann-La Roche eventually bought the PCR and Taq patents from Cetus for $330 million, from which it may have received up to $2 billion in royalties^[7]. In 1989 Science Magazine named Taq polymerase its first "Molecule of the Year". Kary Mullis received the Nobel Prize in 1993, the only one awarded for research performed at a biotechnology company. By the early 1990s the PCR technique with Taq polymerase was being used in many areas, including basic molecular biology research, clinical testing, and forensics. It also began to find a pressing application in direct detection of the HIV virus in AIDS^[8].

References