|
''Taq'' polymerase is a thermostable DNA polymerase named after the thermophilic bacterium ''Thermus aquaticus'' from which it was originally isolated by Chien et al. in 1976. It is often abbreviated to "''Taq'' Pol" (or simply "''Taq''"), and is frequently used in polymerase chain reaction (PCR), a method for greatly amplifying short segments of DNA. ''T. aquaticus'' is a bacterium that lives in hot springs and hydrothermal vents, and ''Taq'' polymerase was identified〔 as an enzyme able to withstand the protein-denaturing conditions (high temperature) required during PCR. Therefore it replaced the DNA polymerase from ''E. coli'' originally used in PCR. ''Taqs optimum temperature for activity is 75–80°C, with a half-life of greater than 2 hours at 92.5°C, 40 minutes at 95°C and 9 minutes at 97.5°C, and can replicate a 1000 base pair strand of DNA in less than 10 seconds at 72°C. One of ''Taqs drawbacks is its lack of 3' to 5' exonuclease proofreading activity〔 resulting in relatively low replication fidelity. Originally its error rate was measured at about 1 in 9,000 nucleotides. The remaining two domains act in coordination, via coupled domain motion. Some thermostable DNA polymerases have been isolated from other thermophilic bacteria and archaea, such as ''Pfu'' DNA polymerase, possessing a proofreading activity, and are being used instead of (or in combination with) ''Taq'' for high-fidelity amplification. ''Taq'' makes DNA products that have A (adenine) overhangs at their 3' ends. This may be useful in TA cloning, whereby a cloning vector (such as a plasmid) that has a T (thymine) 3' overhang is used, which complements with the A overhang of the PCR product, thus enabling ligation of the PCR product into the plasmid vector. ==''Taq'' polymerase in PCR== In the early 1980s, Kary Mullis was working at Cetus Corporation on the application of synthetic DNAs to biotechnology. He was familiar with the use of DNA oligonucleotides as probes for binding to target DNA strands, as well as their use as primers for DNA sequencing and cDNA synthesis. In 1983, he began using two primers, one to hybridize to each strand of a target DNA, and adding DNA polymerase to the reaction. This led to exponential DNA replication, greatly amplifying the amounts of DNA between the primers.〔 However, after each round of replication the mixture needs to be heated above 90°C to denature the newly formed DNA, allowing the strands to separate and act as templates in the next round of amplification. This heating step also inactivates the DNA polymerase that was in use before the discovery of ''Taq'' polymerase, the Klenow fragment of the DNA Polymerase I from ''E. coli''. Use of the thermostable ''Taq'' enables running the PCR at high temperature (~60°C and above), which facilitates high specificity of the primers and reduces the production of unspecific products, such as primer dimer. However, use of the thermostable polymerase eliminates the need for having to add new enzyme to the PCR reaction during the thermocycling process. A single closed tube in a relatively simple machine can be used to carry out the entire process. Thus, the use of ''Taq'' polymerase was the key idea that made PCR applicable to a large variety of molecular biology problems concerning DNA analysis.〔 抄文引用元・出典: フリー百科事典『 ウィキペディア（Wikipedia）』 ■ウィキペディアで「Taq polymerase」の詳細全文を読む スポンサード リンク
|