Definition
The Taq DNA polymerase is a 94 kDa thermostable DNA-dependant DNA polymerase derived from a eubacterium called Thermus aquaticus, whose natural habitat is hot thermal springs (with ambient temperatures of 70–75°C).
Discovery
In 1976, Chien et al.. described a 94 kDa thermostable DNA-dependant DNA polymerase derived from a eubacterium called Thermus aquaticus, whose natural habitat is hot thermal springs (with ambient temperatures of 70–75°C). This thermostable DNA polymerase or “Taq” enzyme was found to possess similar properties to E. coli DNA dependant DNA polymerase I. With its first publication in the mid 1970s, thermo stable DNA polymerase has evolved into a well-known and varied use enzyme. In 1993, its inventor, Kary Mullis, received a Nobel Prize for his research in Taq polymerase .
Structural characteristics
Taq is a stable DNA polymerase with a temperature optimum of 80°C, purified from the extreme thermophile, Thermus aquaticus. The enzyme is free from phosphomonoesterase, phosphodiesterase and single-stranded exonuclease activities. Maximal activity of the enzyme requires all four deoxyribonucleotides and activated calf thymus DNA. The molecular weight of the enzyme was initially estimated by sucrose gradient centrifugation and gel filtrations on Sephadex G-100 to be approximately 63,000 to 68,000. The elevated temperature requirement, small size, and lack of nuclease activity distinguish this polymerase from the DNA polymerases of E coli 1.
Taq Peptides:
Deletion of the first 289 amino acids of the DNA polymerase from Thermus aquaticus (Taq polymerase) removes the 5’ to 3’ exonuclease domain to yield the thermostable Stoffel polymerase fragment. Preliminary N-terminal truncation studies of the Stoffel fragment suggested that removal of an additional 12 amino acids (the Stof?12 mutant) had no significant effect on activity or stability, but that the further truncation of the protein (the Stof? 47, in which 47 amino acids were deleted), resulted in a significant loss of both activity and thermostability. A 33-amino acid synthetic peptide, based on this critical region (i.e., residues 303-335 inclusive), was able to restore 85% of the Stof? 12 activity when added back to the truncated Stof? 47 protein as well as return the temperature optimum to that of the Stof? 12 and Stoffel proteins. Examination of the crystal structure of Taq polymerase shows that residues 302-336 of the enzyme form a three-stranded ß-sheet structure that interacts with the remainder of the protein 3. Photocross-linking of Taq MutS protein to a derivatized heteroduplex DNA containing a 5-IdUrd cross-linking moiety reveals that a region at the NH2 terminus of MutS is closely associated with the major groove of the heteroduplex DNA. Peptide sequencing of the limit trypsin digest of the cross-linked peptide indicates that it maps to residues 25–49, with Phe-39 being the point of cross-linking. Substitution of Ala for Phe-39 results in a mutant protein whose relative affinity for heteroduplex DNA is 3 orders of magnitude lower than that of the wild-type MutS protein. The severe deficiency in DNA binding resulting from a single amino acid change at Phe-39 is not attributable to a gross alteration in the conformation of the mutant protein. The F39A mutant protein is able to dimerize like its wild-type counterpart. In addition, the F39A mutant protein retains thermostability and an ATPase activity that is essentially unchanged from that of the wild-type MutS protein. Taken together, these data strongly implicate the region near Phe-39 as being critical for heteroduplex binding by Taq MutS protein 3.
Mode of Action
PCR is based on Taq DNA polymerase. This enzyme is able to polymerize deoxynucleotide precursors (dNTP) in a temperature range of 75-80° C. A typical PCR reaction is a repetitive series of thermic cycles involving template DNA denaturation, oligonucleotide primer annealing, and extension of the annealed primers by DNA polymerase. This three-step process results in the exponential accumulation of a specific fragment whose termini are defined by the 5' end of the primers. Amplification can be estimated to be 2n, where n is the number of cycles. The first step involves denaturation of double-stranded target DNA by heating the sample to 90-95°C. In the second step, the temperature is lowered to about 5 °C below the melting temperature of the primer, assuring the specificity of the primer annealing and thus the specificity of the product. The third step is carried out by raising the temperature of the sample to 70-73°C, the optimal temperature for primer extension, involving very little denaturation of the enzyme during the 25-30 cycles of a PCR reaction. The primers used are designed on the basis of the known DNA sequence and they must flank the sequence targeted. The choice of the primer sequence is a function of the target and technical requirements, such as a GC content of 50-60%, which gives the optimal annealing temperature of 50-55°C. The molecular composition of the primer must be chosen to prevent the formation of intra-molecular secondary structures and primer dimers. The complementarity between the template and the 3' OH end must be perfect, because Taq DNA polymerase activity is markedly lowered by mismatches and secondary structures. The 5' end can thus modified by extension or base modification without altering the quality of amplification. The yield of the reaction can be modified by the composition of the PCR medium 4.
Functions
Taq polymerase is the most widely used polymerase enzyme in PCR-based methods for the detection of microorganisms in complex biological samples, such as clinical, environmental, and food samples 5.
References
Chien A, Edgar DB, Trela JM (1976). Deoxyribonucleic Acid Polymerase from the Extreme Thermophile Thermus aquaticus. Journal of Bacteriology, 127(3):1550-1557.
Vainshtein I, Malcolm BA, Atrazhev A, Elliott JF, Eom SH, Wishart DS (1996). Peptide rescue of an N-terminal truncation of the stoffel fragment of Taq DNA polymerase. Protein Science, 5:1785-1792.
Malkov VA, Biswas I, Camerini-Otero RD, Hsieh P (1997). Photocross-linking of the NH2-terminal Region of Taq MutS Protein to the Major Groove of a Heteroduplex DNA. The Journal of Biological Chemistry, 272:23811-23817.
Haras D, Amoros JP (1994). [Polymerase chain reaction, cold probes and clinical diagnosis]. Sante.,4(1):43-52.
Al-Soud WA, Rådström P (1998). Capacity of Nine Thermostable DNA Polymerases to Mediate DNA Amplification in the Presence of PCR-Inhibiting Samples. Appl Environ Microbiol., 64(10):3748–3753.
