A "Referral from the Doctor" Blog Article-
When the polymerase chain reaction (PCR) was first described, the Klenow fragment derived from the Escherichia coli DNA Polymerase I was the paramount enzyme for sequence extension. Due to its lack of stability at high temperature, it needs be replenished before each cycle. Upon the discovery of thermophilic bacteria which thrive at temperatures greater than 45 °C, heat-stable polymerases which function at higher temperatures were investigated in an effort to eliminate the need to replenish enzyme following each denaturation cycle.
Taq DNA Polymerase was originally isolated from thermophilic bacterium of the Deinococcus-Thermus group located near the Lower Geyser Basin of Yellowstone National Park by Thomas D. Brock and Hudson Freeze, in 1969. This thriving bacterium was named Thermus aquaticus (T. aquaticus). Several enzymes have been isolated from T. aquaticus, the most known of which is Taq DNA polymerase (Taq). Taq can be isolated either from its original source or from its cloned gene expressed in E. coli. While many similarities in sequence and structure exist between E. coli DNA polymerase I and Taq, differences in enzyme functionality, character and dependencies make Taq the ideal polymerase for use in qPCR-based gene expression analysis, with the exception of sensitive measures of certain bacterial genes. This caveat is explained later.
Characterization of Thermus aquaticus DNA Polymerase
Functionality: Utilizing the inherent 5’ to 3’ exonuclease activity of Taq, researchers are able to simultaneously achieve PCR amplification and signal release from a target-specific fluorogenic probe. The 5’ to 3’ exonuclease activity of Taq cleaves the 5’ terminus of a hybridized oligo probe to release both mono- and oligonucleotides. The probe is hydrolyzed concomitant with strand replication so that the accumulating fluorescent signal correlates with amplification.
Size and activity: While variable weights have been reported, the approximate size of Taq is 94 kd, with the activity of a DNA polymerase localized to the C-terminus and 5’ to 3’ exonuclease activity localized to the N-terminus. To date, no 3’ to 5’ exonuclease activity has been observed as in other polymerases, where the 3’-end mismatched base is excised during a “proofreading” process. The error rate has been reported to be as low as 10-5 for base substitution errors and 10-6 for frameshift errors.
Temperature dependency: Thermophiles are prevalent in nature and certain prokaryotic species thrive at temperatures above 45 °C. The temperature-dependency of Taq makes it optimum at 80 °C, where its catalytic activity is more than ten times that typically observed at 37 °C. It is possible that the decrease in activity above 80 °C is actually due to the denaturation of double-stranded DNA at these temperatures.
Monovalent and divalent cation dependencies: Salt concentrations required for optimum performance are considered to be 40 mM NaCl and 60 mM KCl. Concentrations greater than 100 mM for these monovalent cations are prohibitive to catalytic activity. This is in contrast to the salt-insensitivity of the E. coli Polymerase I enzyme. In addition, Taq may depend — like other polymerases — upon the presence of divalent cations, namely MgCl2 or MnCl2, with optimum concentrations depending on the experimental design. Higher concentrations of manganese can lead to an increased error rate of nucleoside incorporation. Activity with other divalent ions may be significantly decreased or absent, as is the case with greater than 0.25 mM Ca+2. Taq requires the presence of all four species of deoxyribonucleoside triphosphates and DNA for optimum catalytic activity.
pH dependency: The pH optimum for the enzyme is within the range of 7-8 pH units when at 80 °C, and will vary depending on the buffer system used. In a 25 mM Tris-hydrochloride buffer for example, the alkalinity optimum is 7.8 pH units.
The great caveat: uninvited guests
On occasion, cloned Taq polymerase has been shown to have contaminating bacterial DNA that is possibly carried over from the expression vector system or other sources used during polymerase manufacture. This residual contamination may limit the use of cloned Taq in the detection of dilute bacterial DNA in certain samples. Trace contamination may be impossible to completely remove, and indeed certain estimates of contamination counts in commercially available Taq have claimed as many as 1000 genome equivalents of bacterial DNA per unit of enzyme.
Several methods for removing bacterial DNA from Taq polymerase have been tested and appear in the literature. Methods such as exposure to ultraviolet light below 320 nm (UVB or UVC) has the effect of making DNA resistant to amplification; however it also affects the integrity of the Taq polymerase, reducing the efficiency of nucleoside incorporation. Ultra-filtration of the Taq polymerase, while often able to eliminate false-positives, does so with the unwanted effect of decreasing the assay sensitivity. UVA-activated 8-methoxypsoralen treatment to intercalate contaminating DNA into double-stranded DNA is difficult to optimize and inhibits the PCR reaction. In addition to the above methods, restriction endonucleases and DNAse I treatment to digest DNA in Taq preparations may introduce contaminants or become the contaminants themselves.
A recent methodology under interrogation is the serial dilution of the Taq polymerase (up to 32-fold) to effectively dilute-out the contaminating bacterial DNA while maintaining Taq activity and sensitivity. This technique has the consequence of reaction plateauing at lower cycle numbers. This effect generates a lower signal in end-point analysis, with minimal consequence to quantification based on a threshold during the exponential phase.
Written by: Christina Ferrell, Ph.D., Technical Applications Specialist
Detection of specific polymerase chain reaction product utilizing the 5’ – 3’ exonuclease activity of Thermus aquaticus DNA polymerase. 1991. Holland, P. M., Abramson, R.D., Watsohn, R., Gelfand, D.H. Proc. Natl. Acad. Sci. 88: 7276-7280
Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. 1976. Chien, A., Edgar, D.B., Trela, J.M. Journal of Bacteriology. 127 (3): 1550-1557
Characterization of the 5’ to 3’ exonuclease associated with Thermus aquaticus DNA polymerase. 1990. Longley, M.J., Bennett, S.E., Mosbaugh, D.W. Nucleic Acid Research 18(24):7317-7322
Characterization of contaminating DNA in Taq polymerase which occurs during amplification with a primer set for Legionella 5S ribosomal RNA. 1994. Maiwald, M., Ditton, H.J., SonnTaq, H.G., von Knebel Doeberitz, M. Molecular and Cellular Probes 8(1):11-14
Optimizing Taq polymerase concentration for improved signal-to-noise in the broad range detection of low abundance bacteria. 2009. Spangler, R., Goddard, N.L., Thaler, D.S. PLoS ONE 4(9):e7010
High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase. 1990. Eckert, K.A. and Kunkel, T.A. Nucleic Acids Research 18:3739-3744
Comparison of different decontamination methods for reagents to detect low concentrations of bacterial 16S DNA by real-time pcr. 2002. Klaschik, S., Lehmann, L.E., Raadts, A., Hoeft, A., Stuber, F. Molecular Biology 22(3):231-242
Optimization of real-time PCR assay for rapid and sensitive detection of eubacterial 16S ribosomal DNA in platelet concentrates. 2003. Mohammadi,T., Reesink, H.W., Vandenbroucke-Grauls, C.M.J.E., Savelkoul, P.H.M. Journal of Clinical Microbiology 41(10):4796–4798