Nonspecific Detection Systems

A host of detection principles can be used to sense the presence of double-stranded DNA. Fluorescent detection techniques employ dyes that change fluorescence properties upon binding to DNA (this is also discussed in detail in Chapter 2.5).

Fig. 4.2.1. Heterogeneous and homogenous DNA detection.

Interaction of molecules with duplex DNA follows three different binding modes governed by electrostatic interactions, groove binding interactions, or intercalation of planar ring systems between the Watson-Crick base pairs (Box 9). The dyes used for homogeneous DNA-detection typically bind to the minor groove or by intercalation (Figure 4.2.2). Often minor groove-binding dyes have cationic sites that are oriented in space by a crescent-shaped scaffold to fit into the helical curve. A twentyfold increase in fluorescence is observed for the commonly used DAPI dye on binding to duplex DNA [6]. The benzimidazole ring system of the so-called Hoechst dyes enables fluorescence enhancement by a factor of 90 [7]. There are, in addition, numerous proprietary dyes from Molecular Probes.

Intercalator dyes such as ethidium bromide or, more recently, cyanine dyes such as thiazole orange [8] selectively bind to duplex DNA by intercalation between the base pairs (Figure 4.2.2). Dimerization of two fluorophore moieties is a frequently found motif in modern dyes such as TOTO and is performed to increase the binding affinity to DNA [9, 10]. The extended ring systems are constrained into a planar arrangement as a result of intercalation. As a result the re-electrons are effectively delocalized and vivid fluorescence enhancement occurs. This property and the fluorescence increase on minor-groove binding provides a simple means of monitoring the production of duplex DNA during the polymerase chain reaction (PCR). Several platforms are now available for performing real-time PCR analyses and typically a PCR-thermocycler is equipped with a fluorescence detection device [4]. It must be remarked that intercalator dyes bind non-specifically to all duplex-DNA species including primer-dimers and non-specific amplification products. This is a major disadvantage of non-specific detection methods. Melting curve analysis can, however, help in distinguishing specific targets from primer dimers.

0 0

Post a comment