In 1989, Science Magazine named Taq polymerase its first " Molecule of the Year".
Hoffmann-La Roche eventually bought the PCR and Taq patents from Cetus for $330 million, from which it may have received up to $2 billion in royalties. 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. A single closed tube in a relatively simple machine can be used to carry out the entire process. Also, use of a thermostable polymerase eliminates the need to add new enzyme to each round of thermocycling. Use of the thermostable Taq enables running the PCR at high temperature (~60 ☌ and above), which facilitates high specificity of the primers and reduces the production of nonspecific products, such as primer dimer. Taq polymerase is well-suited for this application because it is able to withstand the temperature of 95 ☌ which is required for DNA strand separation without denaturing. This heating step also inactivates the DNA polymerase that was in use before the discovery of Taq polymerase, the Klenow fragment (sourced from E. However, after each round of replication the mixture needs to be heated above 90 ☌ to denature the newly formed DNA, allowing the strands to separate and act as templates in the next round of amplification. This led to exponential DNA replication, greatly amplifying discrete segments of DNA between the primers. In 1983, he began using two primers, one to hybridize to each strand of a target DNA, and adding DNA polymerase to the reaction. 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 the early 1980s, Kary Mullis was working at Cetus Corporation on the application of synthetic DNAs to biotechnology. 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 makes DNA products that have A ( adenine) overhangs at their 3' ends. Fidelity can vary much between Taqs, which has profound effects in downstream sequencing applications. 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. Originally its error rate was measured at about 1 in 9,000 nucleotides. One of Taq's drawbacks is its lack of 3' to 5' exonuclease proofreading activity resulting in relatively low replication fidelity. Interestingly, the common metal ion chelator, EDTA, directly binds to Taq in the absence of these metal ions.
High concentrations of KCl and Mg 2+ inhibit Taq's activity. Taq polymerase is maximally activated at 50mM KCl and just the right concentration of Mg 2+ which is determined by the concentration of nucleoside triphosphates (dNTPs). Small amounts of potassium chloride (KCl) and magnesium ion (Mg 2+) promote Taq's enzymatic activity. Presence of certain ions in the reaction vessel also affects specific activity of the enzyme. At temperatures above 90 ☌, Taq demonstrates very little or no activity at all, but the enzyme itself does not denature and remains intact. A single Taq synthesizes about 60 nucleotides per second at 70 ☌, 24 nucleotides/sec at 55 ☌, 1.5 nucleotides/sec at 37 ☌, and 0.25 nucleotides/sec at 22 ☌. At 75-80 ☌, Taq reaches its optimal polymerization rate of about 150 nucleotides per second per enzyme molecule, and any deviations from the optimal temperature range inhibit the extension rate of the enzyme. Taq's optimum temperature for activity is 75–80 ☌, with a half-life of greater than 2 hours at 92.5 ☌, 40 minutes at 95 ☌ and 9 minutes at 97.5 ☌, and can replicate a 1000 base pair strand of DNA in less than 10 seconds at 72 ☌.
0 kommentar(er)
