what is the special property of taq polymerase that allows it to work in pcr?
Everything You Need to Know almost Taq Polymerase
Taq polymerase is i of the most ubiquitous enzymes in molecular biological science. Since its discovery in 19651, Taq has become the courage of PCR and all the downstream applications that technology enables. This powerful polymerase is the default choice for most amplification and cloning procedures, as well as diagnostic tests, marker gene DNA sequencing, and much more.
What makes Taq polymerase and so special? In today'due south article, we'll have a look at the advantages and disadvantages of Taq, what yous need to get the all-time PCR results from Taq polymerase, and some Taq derivatives that tin can help yous achieve better results for specialised applications.
Why is Taq So Widely Used for PCR?
Taq's popularity benefits from the fact that it was the first high-temperature polymerase discovered by molecular biologists. Merely, that's far from the merely reason it'south yet in such loftier demand today.
Taq polymerase has the important characteristic of being stable at temperatures up to 95°C2. That'due south critical considering this is the temperature at which DNA denatures – a required stride at the get-go of the PCR reaction. While any moderate-temperature polymerase would as well denature at that temperature, rendering information technology useless, Taq remains perfectly set up to outset polymerising your target Dna sample.
On top of that, Taq has the reward of beingness most active in the 70-80°C temperature range2. That's hot plenty that DNA won't reanneal, but primers that take annealed at lower temperatures won't pare off, either. So, Taq polymerase works extremely well at the temperatures about standard PCR reactions require.
Finally, Taq suits basic molecular biology needs extremely well. The polymerase leaves A-tailed products, which tin be easily cloned into a vector using a standard TA cloning kit. Taq is also relatively resilient to Deoxyribonucleic acid sequences with a broad range of GC contents and is not all that picky about target DNA or dNTP concentrations.
What Does Taq Need to Polymerise Finer?
If yous simply added Taq polymerase on its own to a sample of DNA, not much would happen. That'south considering like most enzymes, Taq needs a specialised mix of reagents in order to work properly.
The basic Taq buffer includes potassium chloride, Tris hydrochloride, and Triton X-100. These chemicals are less relevant to optimising your PCR reactions than to keeping Taq safe during storage in the freezer – which itself is important in getting the almost out of your PCRs. While y'all can make your ain buffer to rehydrate and store lyophilized Taq enzyme, the vast majority of polymerase is sold as a master mix with Taq already suspended in the required buffer solution.
More critical to your PCR reactions is magnesium chloride. Magnesium is an essential cofactor for Taq polymerase3. Without it, Taq won't exist able to catalyse the improver of nucleotides to the primer or growing Deoxyribonucleic acid strand. The concentration of magnesium also matters. As more magnesium is added to a PCR reaction, Taq becomes more active, only likewise less specific in polymerising but your target Deoxyribonucleic acid strand.
Because of the concentration-dependent role of magnesium, you can find Taq main mixes with magnesium already included as well as mixes that leave it out. In the latter case, you'll need to add magnesium chloride separately (which requires an extra pipetting step when preparing your PCRs). The advantage is that you can command the exact concentration of magnesium, and so you can experiment to notice the well-nigh optimal blend of Taq productivity and specificity.
What are the Problems with Taq Polymerase?
Taq is widely used for routine PCRs, but unfortunately, it isn't perfect for all applications. The virtually notable drawback to Taq is that it's non the near accurate polymerase out there. Taq has an mistake rate of around one error per 100,000 base pairs4. By comparison, Pfu proofreading polymerase is about 10 times more than authentic. If you're using PCR upstream of cloning or Dna sequencing, that difference in error rate can exist very meaning.
Standard Taq polymerase can also have problems with amplifying target Dna sequences longer than about i kB. Relative to other types of polymerases, Taq has low processivity, which means that its efficiency declines significantly with the length of your amplicon. This depression processivity can be fabricated worse by the presence of PCR inhibitors, which are mutual if you're working with Deoxyribonucleic acid extracted from tissues, plants, or soil.
Finally, while Taq polymerase is most efficient at temperatures above lxx°C, the enzyme continues to piece of work at temperatures less than 50°C. That'southward problematic considering Taq can unintentionally polymerise primers or not-target DNA sequences when the temperature in your PCR reaction falls to permit primers to amalgamate to your target sequence. As a event, Taq is known to produce primer dimers and other unwanted products that can get in the fashion of downstream applications similar cloning.
Specialised Taq Polymerases for Advanced Applications
Thankfully, there are specialised forms of Taq polymerase that solve some of these problems. For instance, highQu ALLin Taq polymerase is a synthetic version of Taq that has more processivity than standard Taq. As a result, you tin more easily accomplish amplicon yields from high-GC templates, templates longer than 1 kB in length, and samples with moderate concentrations of inhibitors.
Another mutual form of Taq, similar the ALLin Hot Start Taq, is engineered with a molecular inhibitor to get in inactive at lower temperatures. Hot start Taq is but functional subsequently heating to 95°C. That dramatically reduces the formation of primer dimers and nonspecific amplicons as a result of polymerisation before the commencement of your PCR reactions. As a result, you tin go higher yields when working with longer target sequences and cut downwardly on background amplicons that normally crave cleanup.
Decision
Taq polymerase has long been the enzyme of option for both routine and complex PCR applications. It perfectly fits the requirements of the PCR reaction, while making downstream applications similar cloning and sequencing uncomplicated. Although standard Taq has some drawbacks, these are largely solved past using an engineered or hot start Taq polymerase. For the vast majority of applications, Taq polymerase remains the go-to enzyme for molecular biologists.
References
one Ishino S, Ishino Y. 2014. Frontiers in Microbiology v:465.
2 Coleman WB, Tsongalis GJ. 2017. Diagnostic Molecular Pathology: A Guide to Applied Molecular Testing.
three Williams M. 2018. Sciencing
4 McInerney, Adams P, Hadi MZ. 2014. Molecular Biology International 287430.
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Source: https://www.clentlifescience.co.uk/everything-taq-polymerase/
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