Table of Contents
What is AmpliTaq Gold DNA polymerase?
Applied Biosystems AmpliTaq Gold and AmpliTaq Gold 360 DNA Polymerases are hot-start enzymes which utilize a chemical modification to inhibit enzyme activity at room temperature.
What are 4 key features of DNA polymerase?
Together, the four properties of DNA polymerases—specificity, thermostability, fidelity, and processivity—make these enzymes highly versatile, and subsequent enhancements further broaden their applications in PCR.
What is the purpose of DNA polymerase isolated from Thermus aquaticus?
Definition. Taq polymerase denotes the heat-stable DNA polymerase extracted from the thermophilic bacteria Thermus aquaticus. It is used to automate the repetitive steps in the polymerase chain reaction (PCR) technique, an extremely important method of amplifying specific DNA sequences.
Is DNA polymerase used in cloning?
Early PCR cloning often used Taq DNA Polymerase to amplify the gene. This results in a PCR product with a single template-independent base addition of an adenine (A) residue to the 3′ end of the PCR product, through the normal action of the polymerase.
What is AmpliTaq?
Applied Biosystems AmpliTaq DNA Polymerase is a recombinant preparation of Taq DNA Polymerase. AmpliTaq DNA Polymerase is used in standard research applications such as routine PCR, genotyping, and colony PCR.
What does Hot Start PCR do?
Hot Start PCR allows for reaction set up at room temperature without non-specific amplification and primer dimer formation. Whereas conventional PCR is often utilized to make exponential copies of your DNA target sequence without an additional temperature-sensitive reaction activation component.
What are the functions of DNA polymerases?
The primary role of DNA polymerases is to accurately and efficiently replicate the genome in order to ensure the maintenance of the genetic information and its faithful transmission through generations.
Why is it important that we use DNA polymerase from an organism like T. aquaticus instead of a human DNA polymerase?
The main reasons that make Thermus aquaticus (Taq) perfect for DNA sequencing are that it’s active across a wide range of temperatures and as such is able to withstand the protein denaturing necessary during PCR so that PCR cycles can be automated, since the polymerase doesn’t need to be added for each cycle.
What is the significance of Thermus aquaticus?
Thermus aquaticus’ proteins are heat-stable. One of them, called Taq DNA polymerase, can keep copying DNA, even after being heated up. This allowed for the production of large quantities of DNA, which started a landslide into the study of our genes. And the rest is history in the making.
What is TOPO TA cloning?
TOPO cloning is a molecular biology technique in which DNA fragments are cloned into specific vectors without the requirement for DNA ligases. Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3′-end of the PCR products.
What is a TA vector?
T vectors are linearized plasmids that have been treated to add T overhangs to match the A overhangs of the PCR product. PCR fragments that contain an A overhang can be directly ligated to these T-tailed plasmid vectors with no need for further enzymatic treatment other than the action of T4 DNA ligase.
What is the benefit of using Taq polymerase in PCR?
Taq DNA Polymerase is highly efficient, so it becomes fully functional as it reaches its optimum temperature. It also has a half-life of more than two hours (at a temperature of 92 °C), a high-amplification capacity, and the ability to add 150 nucleotides per second.
What is the advantage of using Taq polymerase in the PCR reaction?
Advantages of Taq DNA polymerase: Efficiency: It is highly efficient. As it is reached at its optimum temperature, the thermostable polymerase becomes fully functional and adds nucleotides to the growing DNA strand. High amplification capacity: It can insert 150 nucleotides per second during amplification.
What is the temperature of annealing in PCR?
The annealing step (30 sec to 1 min, at temperatures 45–60 °C), is required so that the primers bind to the complementary sequence on each of the DNA single strands. The primers are designed such that they bracket the target of interest and the region of sequence that lies between them is referred to as the amplicon.
What are the three types of DNA polymerase?
Classification. On the basis of sequence similarities, DNA polymerases can fall into three groups: type A, type B and type C, which have homology to polA (pol I), polB (pol II) and polC (pol III) from Escherichia coli, respectively [1,2].
What is amplitaq gold® DNA polymerase?
AmpliTaq Gold® DNA Polymerase is a chemically modified form of AmpliTaq® DNA Polymerase requiring thermal activation. The modified enzyme is provided in an inactive state. Upon thermal activation, the modifier is permanently released, regenerating active enzyme. The resulting hot-start PCR amplification provides increased…
Why upgrade to platinum II Taq hot-start DNA polymerase?
Upgrade to Platinum II Taq Hot-Start DNA Polymerase for universal annealing and a 4X faster cycling protocol. The Applied Biosystems® AmpliTaq Gold® 360 DNA Polymerase, when used with improved AmpliTaq Gold 360 Buffer, and the optional 360 GC Enhancer, amplifies a vast range of DNA sequence contexts.
Why is high-temperature incubation required to activate amplitaq gold DNA polymerase?
A high-temperature incubation step is required to activate AmpliTaq Gold DNA Polymerase, which ensures that the active enzyme is generated only at temperatures in which the DNA is fully denatured and when the primers are not annealed.
What are the different types of amplitaq DNA polymerase?
AmpliTaq DNA Polymerase is available in different formulations as summarized in the table below. AmpliTaq 360 DNA Polymerase and AmpliTaq LD DNA Polymerase are ultra-pure preparations that are used for experiments where it is important to reduce the incidence of false positives.