Double-stranded DNA binding dyes

More recently double stranded DNA-binding dyes, (e.g., SYBR Green), have been introduced (Giulietti et al. 2001) which removed the need for an expensive, specific probe to be designed. Other sophisticated tools have been developed to work in conjunction with the Taqman method, for example molecular beacons, scorpions and hybridisation probes. These techniques rely on the FRET (Fluorescence Resonance Energy Transfer) principle but do not require the nuclease activity of the Taq polymerase. The different real-time... 

The ability of some fluorescent dyes to bind DNA quantitatively is exploited in flow cytometry to determine the DNA content of a cell. Dyes such as propidium iodide that bind double-stranded DNA stoichiometrically can be used for the purpose. The intensity of red fluorescence is directly related to the amount of DNA bound by propidium iodide. By comparing the fluorescence intensity of the test specimen and, in turn, its DNA content to the fluorescence intensity of specimens containing normal diploid amounts of DNA, a DNA histogram can be generated. By computing a DNA index, which is the ratio of DNA content of a test specimen to the DNA content of a specimen containing a normal diploid population, information related to the presence of an aneuploid tumor population can be obtained. The DNA index of 1 would imply that the DNA in the test specimen is from a normal diploid population (2N DNA), whereas the DNA index of an aneuploid population will be greater or less than 1. Thus, the DNA index of a tetraploid (4N DNA) would be 2. 

This protocol uses propidium iodide (PI) as the fluorescent tracer for DNA content (3-6). PI binds to both double-stranded DNA and double-stranded RNA. Therefore, RNase will be used to reduce the double-stranded RNA resulting in only DNA staining. For alternative DNA-specific ligands and dyes, see Chapter 30. 

Much attention has been focussed lately on the family of asymmetric cyanine dyes for use in fluorescence detection of nucleic acids. These dyes show a significant enhancement in fluorescence intensity (100- to 1000-fold) upon binding to double-stranded DNA as compared to that from the fluorophore in solution. Use of cyanine fluorophores may be advantageous for use in assay design and sensor applications with respect to some of the more commonplace dyes, such as ethidium bromide and Hoechst 33342, as these latter dyes exhibit significant fluorescence intensity as background when in solution and have significantly lower enhancement in emission intensity [42]. 

Perhaps the most well-recognized fluorescent dye for detection of DNA hybridization is ethidium bromide (EtBr). EtBr is a cationic phenanthridinium compound that can bind to DNA by intercalation. This dye has an excitation maxima at 518 nm when bound to double-stranded DNA (dsDNA). Excitation of EtBr is often done by use of an argon ion laser, making this fluorophore a viable choice for applications in optical sensors as well as confocal scanning laser microscopy and fluorometry [41]. The structure of ethidium bromide is shown in Fig. 6. 

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). 

Lower nucleic acid concentrations are best determined fluorimetrically. These methods generally depend on the fact that certain dyes can bind to nucleic acids by intercalating between successive base pairs, and this binding is accompanied by marked increases in the fluorescence quantum yield. Ethidium bromide fluorescence (Aex 260-360 nm Af.rr] 560 nm), which is commonly used to visualise nucleic acids in gel electrophoresis, can also be used to quantitate double stranded DNA and RNA with a sensitivity of about 10 ng (Karsten and Wollenberger 1977). The dye 4,6-diamidino-2-phenylindole (DAPI) (Aex 360 nm 2f.m 450 nm) can be used to quantitate DNA specifically with a detection limit of about lng (Brunk et al. 1979). 

When free in aqueous solution, these dyes exhibit limited fluorescence, but upon binding DNA, their fluorescence increases markedly. Ethidium binds to RNA and DNA while DAPI and Hoechst 33258 selectively interact with DNA. Hoechst 33258 at a final concentration of 1 pg/mL in 0.05 M phosphate, pH 7.4, with 2 M NaCl has been used to quantitate as little as 10-ng double-stranded DNA.18 Because these dye-binding methods involve intercalation between the bases of double-stranded DNA they are not useful for the quantitation of single-stranded DNA. 

Fluorescence Increases as dye binds to double-stranded DNA product... 

In general, small molecules that bind to double-stranded DNA can be divided into two classes (1) intercalators, and (2) minor-groove binders. DNA binding dyes such as SybrGreen I are cheap and easy to use. Therefore, SybrGreen I is the common choice for optimizing real- time PCR. When free in solution, SybrGreen I displays only a bit... 

The binding dye is a small molecule that fluoresces upon binding to double-stranded DNA. As PCR amplification produces more and more dsDNA molecules, the fluorescence signal increases (Fig. 6.6). Dyes are classified into two categories... 

Detection of SYBR Green I Fluorescence The SYBR Green I is a simple, inexpensive and sensitive method. SYBR Green I is an intercalating dye that binds to the minor groove of double stranded DNA (dsDNA) molecules, regardless its sequence. After binding the dye to the dsDNA, the intensity of the fluorescence emission increases more than 300-fold, which provide excellent sensitivity to quantify the dsDNA... 

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