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PCR Polymerases - PCR ENZYMES

Choosing the right DNA polymerase is determined by the aims of the experiment. There is a vast amount of commercially available anzymes to choose from that differ in their processivity, thermal stability and fidelity. The most common enzyme used is Taq DNA polymerase also known as commercially as, AmpliTaq® DNA polymerase.

AmpliTaq DNA Polymerase

AmpliTaq DNA Polymerase is a highly characterized recombinant enzyme for PCR. Produced in E. coli from the Taq DNA polymerase gene, thus assuring great purity. It is commonly supplied and used as a 5 U/μL solution in buffered 50% (v/v) glycerol.

Physical Properities: The enzyme is a 94 kDa protein with a 5’-3’ polymerization activity that is most efficient in the 70°–80°C range. It is incredibly thermostable, with a a half-life at 95°C of 35–40 min. In terms of thermal cycling, the half-life is approx 100 Cycles. PCR products using this enzyme will have single base overhangs on the 3’ ends of each polymerized strand (this can be exploited for use with T/A cloning vectors).

Bio Reactions: DNA polymerase requires magnesium ion as a cofactor and catalyzes the extension reaction of a primed template at 72°C. The four dNTPs are used to extend the primer and therby to copy the target sequence. Also modified nucleotides can be incorporated into PCR products.

Activities: This enzyme has a fork-like structure dependent, polymerization enhanced, 5’-3’ nuclease activity. The activity that it possesses allows it ti degrade downstream primers and indicated that circuka targets should be linearized before amplification. Also this nuclease activity has been employed in a fluorescent signal-generating technique for PCR quantitation. This enzyme does not have an inherent 3’-5’ exonuclease or proofreading activity, but does produce amplicons of high yield for most applications.

AmpliTaq Gold Polymerase

This enzyme is a chemically modified AmpliTaq DNA polymerase, The reversible modification keeps the enzyme inactive at room temperature. High temperature and low pH promote the reversal, restoring the enzyme activity. These conditions occur in a Tris-buffered PCR at 92°–95°C (Tris-Cl formulated to pH 8.3 at 25°C drops below pH 7.0 above 90°C). AmpliTaq Gold is formulated to perform the same as 5 U/μL AmpliTaq DNA polymerase. Therefore, a hot start can be added to most PCRs optimized with AmpliTaq DNA polymerase by substituting AmpliTaq Gold and adding a 10-min, 95°C, pre-PCR, activation step.

The same results can be achieved without the pre-PCR activation step by adding an additional 10 or more PCR cycles. Under these conditions, the enzyme is activated incrementally during the PCR denaturation steps.

Also see PCR Polymerases an excellent article at PCR Station.

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