Alternative Scenarios for Assaying Polymerase Activity

Several approaches have been reported for the screening of polymerase activity, for example radioisotope assays such as scintillation proximity assays [56, 57] or fluorescence-based assays [58-61]. Most of these assays, however, suffer from use of tedious procedures, the use of radioisotopes, or use of expensive reagents for fluorescence signal generation. A convenient means of online monitoring of DNA polymerase activity has recently been presented by Andreas Marx and Daniel Summerer [62]. Their technique involves a DNA template that forms a stable hairpin structure labeled at two positions:

1. internally at the origin of the hairpin stem (fluorescent dye: carboxyfluorescein, FAM), and

2. at the 5'-terminus (fluorescent dye: TAMRA).

Because of the hairpin formation, these dyes are in such a close proximity that their fluorescence is quenched (molecular beacon; Box 18) unless the structure is unfolded in the course of second-strand synthesis (Figure 4.3.4b). Thus, detection of a fluorescence signal from one of both dyes is a direct measure of the progress of the reaction. These researchers also showed that primer extension reactions can be monitored directly in cleared lysates of cells overexpressing the Klenow fragment of E. coli DNA polymerase I. Thus, the ''molecular beacon assay'' might supersede extensive purification.

Further promising polymerase assays employ fluorescence polarization (a property that evaluates the rotational diffusion of fluorophores, which is related to the molecular volume), or the detection of fluorescence intensity during incorporation of fluorescence-labeled substrates (Figure 4.3.4a).

Fig. 4.3.4. Alternative schemes for (general) assessment of polymerase activity. (a) Active polymerases elongate the primer-template, thereby randomly incorporating fluorescence-labeled nucleotides (at a low density). The reaction progress can be monitored either by determination of the fluorescence intensity, or by using fluorescence techniques that also

Fig. 4.3.4. Alternative schemes for (general) assessment of polymerase activity. (a) Active polymerases elongate the primer-template, thereby randomly incorporating fluorescence-labeled nucleotides (at a low density). The reaction progress can be monitored either by determination of the fluorescence intensity, or by using fluorescence techniques that also evaluate the product lengths (for example, by determining the velocity of translational or rotational diffusion; these values are related to the molecular volume, or mass). (b) A fluorescence signal arises if the dually labeled probe, a molecular beacon, opens during elongation of the primer-template [62].

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