Taq DNA Polymerase (Native and Recombinant)
Invitrogen sells the native form of the Taq DNA polymerase and a cloned version that is expressed in E. coli. Both will leave a 3' dA overhang on ~30% of the ends of PCR fragments).
Taq DNA Polymerase catalyzes the incorporation of dNTPs into DNA. It requires a DNA template, a primer terminus, and the divalent cation Mg++. Taq DNA Polymerase contains a polymerization dependent 5'-3' exonuclease activity. It does not have a 3'-5' exonuclease and thus no proof reading function. Despite this, the enzyme synthesizes DNA in vitro with reasonable fidelity.
In repeated use for cycle sequencing, it has shown no tendency to misincorporate nucleotides. The recombinant Taq DNA Polymerase expressed in E. coli shows identical characteristics to native Taq from Thermus aquaticus with respect to activity, specificity, thermostability and performance in PCR.
Taq DNA Polymerase is supplied with a 10X Taq Reaction buffer (200 mM Tris pH 8.4, 500 mM KCl) and a tube of 50 mM MgCl2. The buffer/MgCl2 is also sold separately.
- PCR, 3' RACE, 5' RACE. Taq is stable up to 95°C; thus it is not necessary to replenish the enzyme in a 35 cycle PCR . The maximal enzyme activity is between 70-75°C which minimizes secondary structure of the template and results in high polymerization yield. Annealing temps can be chosen from 30-70°C. Taq can be used to generate PCR products.
- Cycle sequencing. It gives more uniform bands than Klenow. The high temperature extension and cycling permits difficult sequences (hairpins) to be sequenced more readily. It can read through homopolymer sequences (i.e. dG:dC) better than Klenow. [Murray(1989)NAR 17, 8889].
- Run Off Synthesis (i.e. Cycle Labeling). Making high specific activity single strand specific probes. [Sturzl(1990)Anal. Bioch. 185, 164]. Cycle Labeling can theoretically be done with biotinylated nucleotides [Finckh (1991) BioTechniques 10.1, 35].
Reaction Parameters & Enzyme Details
Taq DNA Polymerase is a 832 amino acid single subunit enzyme with a MW of 94,000. The amino acid sequence for the enzyme can be found in [Lawyer(1989)JBC 264, 6427]. It probably does not contain disulfide bridges since the unreduced enzyme has the same mobility in SDSgels as the reduced enzyme.
|Taq Polymerase Half Life||Temperature|
|Optimum pH||Active from pH 7.5 - 9.5|
|Divalent Cation||1-5 mM Mg++, 2 mM Optimum|
|Salt Effects||Stimulated by increasing KCl up to 50 mM. NaCl does not show this effect.|
|Temperature||Relative rates: 72°C = 100%, R.T. = 0.4%, 37°C = 2.55, 55°C = 40%, 85°C = 40%|
|Rate||Very processive enzyme. It has an extension rate of 35-100 nt/sec @ 75°C [Wittwer (1991)BioTechniques 10(1), 76.|
|Inhibitors||Bromophenol Blue [Wittwer(1991) BioTechniques 10.1, 82]. Urea, DMSO DMF, formamide and SDS all inhibit Taq to some extent. KCl at concentrations greater than 75 mM.|
|Compatability||Xylene Cyanol (0.02%), Tartrazine and Ficoll (2.5%) do not inhibit this enzyme.|
|Km for DNA||1.5 nM|
|Effect of DMSO||Taq is inhibited 47% at a concentration of 10% DMSO. It can still be used for PCR & Cycle Sequencing of GC rich DNA in the presence of 10% DMSO if the amount of Taq is doubled. Sun(1993)BioTechniques 15.3, 372.|
|Reaction Rate||~35-150 nts/second|
The error rate reported for Taq is very much dependent on the assay used. Different errror rates reported in the literature (and the corresponding reference citation are listed above).
|1.1 x 10-4 base substitutions/bp||Tindall, et. al. (1988) Biochemistry 27, 6008. Assay = Reversion of Opal Suppression in LacZ.|
|2.4 x 10-5 frameshift mutations/bp||Tindall, et. al. (1988) Biochemistry 27, 6008.|
|2.1 x 10-4 errors/bp||Keohavong, et. al. (1989) PNAS 86, 9253. Assay = Denaturing Gradient Gel Electrophoresis.|
|7.2 x 10-5 errors/bp||Ling, et. al. (1991) PCR Methods Appl 1(1), 63.|
|8.9 x 10-5 errors/bp||Cariello, et. al. (1991) Nucleic Acids Research 19(15), 4193. Assay = DGGE|
|2.0 x 10-5 errors/bp||Lundberg, et. al. (1991) Gene 108, 1. Assay = Loss of LacI function.|
|1.1 x 10-4 errors/bp||Barnes, et. al. (1992) Gene 112, 29. Assay = Loss of LacZ function.|
"Errors/bp" indicates total errors (base substitutions, frameshifts, etc).