Taq polymerase /ˌtæk ˈpɒlɨməreɪz/ is a thermostable DNA polymerase named after
the thermophilic bacterium Thermus aquaticus from which it was originally
isolated by Chien et al. in 1976. It is often abbreviated to "Taq Pol", and is
frequently used in polymerase chain reaction, a method for greatly
amplifying short segments of DNA. T. aquaticus is a bacterium that lives
in hot springs and hydrothermal vents, and Taq polymerase was identified as an
enzyme able to withstand the protein-denaturing conditions required
during PCR. Therefore it replaced the DNA polymerase from E. coli originally
used in PCR. Taq's optimum temperature for activity is 75–80°C, with a
half-life of greater than 2 hours at 92.5°C, 40 minutes at 95°C and 9 minutes
at 97.5°C, and can replicate a 1000 base pair strand of DNA in less than 10
seconds at 72°C. One of Taq's drawbacks is its lack of 3'
to 5' exonuclease proofreading activity resulting in relatively low replication
fidelity. Originally its error rate was measured at about 1 in 9,000
nucleotides. The remaining two domains act in coordination, via coupled domain
motion. 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 Taq for high-fidelity
amplification. Taq makes DNA products that have A
overhangs at their 3' ends. This may be useful in TA cloning, whereby a cloning
vector that has a T 3' overhang is used, 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, greatly amplifying the amounts of DNA between the primers.
However, after each round of replication the mixture needs to be heated above
90°C to denature the newly formed DNA, allowing the strands to separate and act
as templates in the next round of amplification. This heating step also
inactivates the DNA polymerase that was in use before the discovery of Taq
polymerase, the Klenow fragment of the DNA Polymerase I from E. coli.
Use of the thermostable Taq enables running the PCR at high temperature,
which facilitates high specificity of the primers and reduces the production
of unspecific products, such as primer dimer. However, use of the thermostable
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.
Patent issues 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. 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 in AIDS. In December 1999, U.S. District Judge
Vaughn Walker ruled that the 1990 patent involving Taq polymerase was issued, in
part, on misleading information and false claims by scientists with Cetus
Corporation. The ruling supported a challenge by Promega Corporation against
Hoffman-La Roche, which purchased the Taq patents in 1991. Judge Walker cited
previous discoveries by other laboratories, including the laboratory
of Professor John Trela in the University of Cincinnati department of
biological sciences, as the basis for the ruling.
Protein domain = Function =
In molecular biology, this protein domain, Taq polymerase exonuclease,
refers to a domain found in prokaryotic Taq DNA polymerase.
= Structure = This domain where it assumes a
ribonuclease H-like motif. The domain confers 5'-3' exonuclease activity to
the polymerase. See also
Biological machines DNA polymerase I
DNA replication Enzyme catalysis
Protein domain dynamics References