Taq DNA polymerase

Taq DNA polymerase:-


  Taq DNA polymerase is a DNA dependent DNA polymerase, first isolated from the hot spring bacterium, Thermus aquatics in 1976 and in 1989. Due to its wide use in molecular biology (primarily PCR), it is termed as ‘Molecule of the Year’. This thermophilic DNA polymerase encodes an 832-amino acid, 94 kDa protein, which consists of two domains.

1. -NH2 domain:  Similar to 5’-3’ exonuclease domain of members of polymerase I family of DNA polymerase

2. -C terminal domain contains a catalytically inactive 3’-5’ exonuclease and a polymerase sub domain, similar to klenow of DNA polymerase I.

The thermal stability of Taq DNA polymerase is attributed to its hydrophobic core and stable electrostatic interactions and high density of proline residues on the surface of the enzyme.

The optimal activity is at 75-800C temperature and at 600C, the activity is reduced by a factor of 2 and at 370C, its activity is reduced to only 10%.

To initiate DNA synthesis, like other DNA polymerases, it also requires a primer that is annealed to the template strand and caries an extensible 3’-OH group.

Taq DNA polymerase requires Mg2+ for its optimal activity. Phosphate buffers inhibit Taq DNA polymerase and therefore should be avoided. The reaction is usually carried out in the presence of Tris buffer at pH 8.3. Because of the lack of a proofreading function, the rate of misincorporation of dNTPs is high in PCR reactions which are catalyzed by Taq polymerase (or any other DNA polymerase that does not have editing domains).
Several mutant forms of native polymerases, are also available like Pfu And vent. Both of them have proofreading activities contributed by 3’-5’ exonuclease.

 Pfu polymerase is therefore known to generate lowest errors while vent is probably intermediate between Taq and Pfu.

 Pfu polymerase is isolated from Pyrococus furiosus Vent is isolated from Thermococcus litoralis (also known as Tli polymerase) 

What is the Conditions and requirements that influence Enzyme Activity for the use of Restriction Endonuclease at the time of treatment with plasmid ?

What is the Conditions and requirements that influence Enzyme Activity for the use of Restriction Endonuclease at the time of treatment with plasmid ?

One unit activity :-

it is defined as amount of enzyme required to digest 1µg of reference DNA in 60 minutes at 370C. Usually reference DNA is λ phage DNA.

Buffer:- 

The most critical determinant of the enzyme activity is the ionic concentration (NaCl content) of the buffer. There are commercially 3 buffer systems available for the activity, low salt buffer (20mM), medium (100mM) or high (250mM) NaCl buffers. The buffer is usually 10mM Tris-HCl, pH 8.0, supplemented with Magnesium salt (often 50mM MgCl2), a reducing agent (usually 1mM DTT), and some enzymes require BSA (100µg/ml) and salt (NaCl). The reaction does not require ATP.

Quality of DNA :-

Preparation of DNA that has to be cleaved should be free of contaminants such as phenol, CHCl3, alcohol, EDTA, detergents, excessive salts, protein, RNA, all of which can pose a hindrance to the enzyme’s activity.

Stopping a Reaction :

To terminate Restriction enzymes activity, heat inactivation is a way but for the enzymes which it does not work, phenol / CHCl3 extraction is another means of inactivation of Restriction enzymes.

Incubation Temperature and Time  : - 

Recommended temperature for most of the Restriction enzymes is 370C except for the ones which are isolated from thermophilic bacteria which require higher temperature ranging from 50-650C. While some restriction enzymes have short ½ lives at 370C and for those lower temperatures are required. Incubation time as per the unit definition is 1 hour but it can be manipulated. Example, it may be shortened if more units of Restriction enzyme is added or longer times are used to allow a reaction to proceed to completion with fewer enzymes units.