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Polymerases from NEB

The ability to copy genetic material has elevated DNA polymerases to a prominent role in modern biology. Arthur Kornberg's discovery and characterization of E. coli DNA Polymerase I in 1955 was the first step in the evolution of this class of enzymes into powerful tools for the study and manipulation of genes. Subsequent investigation has proven that their utility extends far beyond DNA synthesis. Currently, NEB supplies DNA polymerases differing in properties such as temperature preference, proofreading exonuclease activity, processivity and strand displacement. Each of these parameters will factor into the choice of a polymerase. In order to facilitate matching the ideal DNA polymerase to a particular research application, the chart below and accompanying text describes properties associated with specific enzymes.

Thermostability: DNA is a dynamic molecule whose structure is stabilized by a large number of weak interactions. The stability of the DNA double helix depends on a variety of factors, including DNA sequence, pH, ionic strength, solvents and temperature. In particular, as the temperature is increased, the weak interactions are sequentially disrupted, first resulting in localized denaturation of the terminal and selected internal sequences, and finally in complete separation of DNA strands. The degree to which this destabilization is desired or tolerated depends on the application. Consequently, it is important to match the thermal characteristics of DNA polymerases to desired outcomes.

For example, cloning procedures such as end-polishing are maximized when the termini are stabilized, suggesting use of a polymerase derived from a mesophilic organism. Second strand cDNA synthesis and nick-translation are other applications that have traditionally used mesophilic enzymes. Such enzymes are maximally active at temperatures of 25–40°C and retain signifi-cant activity at even lower temperatures. They commonly can be heat-inactivated and work in the same buffers used by restriction endonucleases and ligases, obviating the need for subsequent DNA purification.

A variety of other molecular biology applications require high temperature to denature the DNA prior to primer annealing or during polymerization to reduce secondary structure, thus reducing polymerase pausing. Archaeal DNA polymerases such as Vent_R® and 9°N™ are derived from hyperthermophiles and are extremely resistant to inactivation even at 100°C and display maximal polymerase activity at 75-85°C. Bacterial thermophiles have yielded enzymes such as Taq and Bst DNA polymerases, which have similar polymerization temperature optima, but somewhat reduced stability at 95°C when compared with archaeal counterparts.

Proofreading: Another necessary consideration with respect to DNA polymerase function is whether the reaction will benefit from the presence of a 3´->5´ proofreading exonuclease moiety. The proofreading mechanism is intrinsic to most DNA polymerases and allows the enzyme to check each nucleotide during DNA synthesis, and excise mismatched nucleotides in the 3´ to 5´ direction. The proofreading domain also enables a polymerase to remove unpaired 3´ overhanging nucleotides to create blunt ends. Protocols such as high-fidelity PCR, 3´ overhang polishing and second strand synthesis require the presence of a 3´->5´ exonuclease.

In contrast, some applications are enhanced by use of polymer-ases without proofreading activity. For example, the efficiency of DNA labeling is enhanced by the absence of proofreading be-cause it prevents excise of incorporated bases, allowing for the use of less of the modified base. Modified base incorporation assays such as multicolor analysis of gene expression, gene mapping, and in situ hybridization, which utilize DNA that has been labeled with a fluorescent nucleotide to facilitate detection, are well matched to NEB’s exonuclease-deficient DNA polymer-ases. Non-proofreading polymerases are also indispensable when partially filling in 5´ overhangs with only selected dNTPs. Addition of an untemplated da residue at the 3´ terminus of blunt ends, a requirement for TA cloning, is also promoted by non-proofreading enzymes. In addition to several wild type polymerases in each of these categories, NEB offers genetically altered versions of several proofreading polymerases where the proofreading exonuclease has been attenuated or abolished.

Strand Displacement: The term strand displacement describes the ability to displace downstream DNA encountered during synthesis. Protocols such as the isothermal amplification method Strand Displace-ment Amplification (SDA) exploit this activity. NEB produces DNA polymerases with varying degrees of strand displacement activity as well as a few whose ability to strand displace is temperature dependent. Polymerases lacking strand displace-ment activity are used in gap-filling reactions such as those employed in site-directed mutagenesis protocols.

In contrast to strand displacement, some polymerases degrade an encountered downstream strand via a 5´->3´ exonuclease activity. This activity is employed for nick-translation protocols.