Taq polymerase
| Taq polymerase I. | ||
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Existing structural data: s. UniProt |
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| Mass / length primary structure | 835 amino acids | |
| Identifier | ||
| Gene name (s) | polA | |
| External IDs | ||
| Enzyme classification | ||
| EC, category | 2.7.7.7 , nucleotidyl transferase | |
| Substrate | Deoxyribonucleoside triphosphate + DNA n | |
| Products | Diphosphate + DNA n + 1 | |
The Taq polymerase is thermostable DNA polymerase of the bacterium Thermus aquaticus ( Taq ). This bacterium lives in geysers at around 70 ° C. Taq polymerase is extremely heat-resistant and belongs to the extremozymes . In 1969 the Taq polymerase was first isolated by Thomas D. Brock and Hudson Freeze .
properties
The enzyme is mainly used for DNA amplification using the polymerase chain reaction . DNA polymerases from mesophilic organisms would be denatured if heated during the polymerase chain reaction and would have to be added after each cycle. This is not necessary for DNA replication with Taq polymerase, as the enzyme is still very stable even at high temperatures (the enzyme half-life is approx. 9 minutes at 97.5 ° C). However, DNA amplification with Taq is error-prone, because the enzyme has no 3 '→ 5' exonuclease activity and therefore no proof reading function. The error rate of Taq polymerase is 8 · 10 −6 errors per base pair . In the amplified DNA fragments, mutations are therefore often found that arise from inaccurate copying of the template strand. If sequence-exact DNA amplificates are required, the use of polymerases with a proof reading function, such as Pfu , Pwo polymerase or Pfx polymerases (which were originally also isolated from thermophilic microorganisms) is recommended , but their use is more costly.
When amplifying a DNA fragment, the Taq polymerase attaches an additional nucleotide (dATP) to the 3 'end of the synthesized strand. One speaks of a 3'-A overhang (A for adenine ), since there is no complementary nucleobase ( thymine ) on the template strand . The 3 'overhang is not corrected by the lack of the proof reading function and is used in the TA cloning process . The Stoffel fragment is created by deleting the first 289 amino acids .
The enzyme is mass-produced by transferring the gene to a strain of the Escherichia coli bacterium . The protein is purified from these cultures and stored in a glycerol-containing buffer at −20 ° C.
Individual evidence
- ↑ FC Lawyer, S. Stoffel, RK Saiki et al .: High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5 'to 3' exonuclease activity . In: PCR Methods Appl. tape 2 , no. 4 , May 1993, pp. 275-287 , PMID 8324500 .
- ↑ J. Cline, JC Braman, HH Hogrefe: PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases. In: Nucleic Acids Research . (1996), Vol. 24 (18), pp. 3546-51. PMID 8836181 ; PMC 146123 (free full text).
- ↑ S. Dabrowski, J. Kur: Recombinant His-tagged DNA polymerase. II. Cloning and purification of Thermus aquaticus recombinant DNA polymerase (Stoffel fragment). In: Acta biochimica Polonica. Volume 45, Number 3, 1998, pp. 661-667, PMID 9918492 .