TAQ POLYMERASE-THE THERMOSTABLE DNA POLYMERASE
The progress in the discipline of molecular biology and evolution
of recombinant DNA Technology is starred by a number of landmark
experiments and path-breaking discoveries.
One such phenomenal invention is that of the PCR or the
Polymerase Chain Reaction technique.
PCR is a technique used to rapidly amplify a gene or a segment of
a gene using repeated cycles of in vitro DNA synthesis.
When the polymerase chain reaction (PCR) was originally
developed by an American scientist Kary Mulis and his coworkers in
1983, the only enzyme which was being used for in vitro DNA synthesis
and sequence extension was the Klenow fragment derived from the E.
coli DNA Polymerase I.
But, like most other known enzymes at that time, the Klenow was
a thermolabile enzyme that got denatured at the elevated temperatures
required for PCR .
Hence there was a need to refill the enzyme before each replication
and extension cycle which made the process tedious and cumbersome.
This problem was resolved only with the knowledge and
application of the thermostable DNA polymerase.
The first major breakthrough in this direction had come earlier when
Thomas Brock and Hudson Freeze (1969) discovered a thermophilic
archaebacterium thriving at extremely elevated temperatures in the hot
springs and thermal vents in the Yellowstone National Park, USA and
named it Thermus aquaticus (T. aquaticus).
Rcently, several thermostable enzymes were purified from T.
aquaticus.
Chien and his coworkers (1976) isolated and characterized one of
the first thermostable DNA polymerase from the T. aquaticus which was
shown to retain its functional activity at elevated temperatures they
named as the Taq polymerase.
Taq polymerase was one of the first thermostable DNA
polymerase that was successfully used to eliminate the need to replenish
enzyme after each denaturation cycle in PCR.
Randall Saiki was the first scientist to employ the thermostable
Taq polymerase for in vitro DNA synthesis by polymerase chain
reaction.
It was established that the Taq polymerase retained its functional
activity at above average survival temperatures and that its half-life was
nearly 40 minutes at 95C and nearly 5 minutes at 100C.
This feature allowed the use of Taq polymerase as the in vitro
DNA synthesizing enzyme for PCR protocols.
Enzyme Properties
The extensive use of Taq polymerase as the enzyme of choice for
routine PCR technique may be attributed to its exceptional thermal
stability and high resistance to denaturation at elevated
temperatures.
The structural and functional properties of Taq polymerase have
been illustrated and documented through series investigative
studies including cloning, overexpression, crystallography etc.
While Taq pol shows certain striking similarities with E. coli DNA
pol I in terms of sequence and structure, yet, Taq has certain
unique features in terms of enzyme functionality, character and
dependencies which differentiate Taq pol from Pol I and make Taq
the ideal polymerase for use in PCR and qPCR-based gene
expression analysis.
Domain Structure, Function and Fidelity
Cloning and characterization of Taq pol has established close
resemblance of Taq pol with E. coli Pol I. These two polymerases
show similar domain structure and organization in both the
primary and the tertiary structural forms.
Taq pol is also a single subunit DNA dependent DNA
polymerase enzyme that has a full length of 832 amino acids and
molecular weight of 94 kDa.
Taq pol in its purified full length form shows a single subunit
structure with two distinct domains exhibiting distinct functions.
Similar to DNA pol I, the C-terminal of Taq is associated with the
5′to3′ polymerase activity while the Nterminal domain of Taq
exhibits 5′to 3′ exonuclease activity.
Taq polymerase altogether lacks the 3′to 5′ exonuclease
(proofreading) function associated with the central domain of E.
coli. DNA pol I. Thus Taq pol performs DNA polymerization with
a relatively moderate reliability as it is not equipped to excise
polymerization errors incurred during synthesis.
Functionally, Taq polymerase can synthesize DNA in 5’to 3’
direction only and it lacks the ability of de novo DNA synthesis;
thus it requires a DNA template and a primer with a free 3’-OH
terminus where it can continue strand extension and achieve DNA
templated DNA synthesis.
Taq DNA polymerase exhibits a modest fidelity and is reported to
polymerize dNTPs with an error rate between 1 × 10−4 and 2 ×
10−5 errors per base pair, depending on experimental conditions.
In routine PCR experiments, Taq is known to incur 1 error in 9000
nucleotides.
All these properties together account for the fact that Taq pol is the
most obvious choice for in vitro analysis of DNA sequences and is
frequently used to detect specific in vivo mutations.
Mode of Action
Taq polymerase has been well characterized and its crystal
structure has been elucidated by X-ray crystallography.
Taq polymerase, in its three dimensional tertiary structure
reportedly resembles the conventional “human right hand cupped
structure”, a property that is common and typical for all the known
DNA polymerases characterized so far.
Crystal structure analyses reveal that the site for binding the single
stranded-template is located in the subdomains corresponding to
the fingers, binding site for the incoming dNTP is associated with
the subdomain corresponding to the palm like region and the
thumb shaped subdomain hosts the binding site for the dsDNA.
Further, it is also reported that the polymerase subdomains of Taq
pol shows high degree of homology with that of the Klenow
fragment and have 51% amino acid similarity.
A significant deviation of the Taq from the Klenow fragment is in
the fact that the 3’-5’ exonuclease domain of Taq is inactive.
