DNA beacons

Most real-time PCR kits are based on such DNA beacons, which npon replication are incorporated into the newly formed strand, or conversely, digested by DNA polymerases, in both cases generating a flnorescence signal in dependence on the target DNA concentration. 

Real-time PCR is a quantitative method for measuring amplicons as they are produced by measuring the increase in fluorescence of a dye added to the reaction mixture.12,104,105 Methods using fluorescent reporters, such as SYBR Green,104,106 TaqMan ,107,108 or molecular beacons,9 collect quantitative data at the time when DNA is in the exponential phase of amplification. 

Hwang GT, Seo YJ, Kim BH (2004) A Highly discriminating quencher-free molecular beacon for probing DNA. J Am Chem Soc 126 6528-6529... 

Fujimoto K, Shimizu H, Inouye M (2004) Unambiguous detection of target DNAs by excimer—monomer switching molecular beacons. J Org Chem 69 3271-3275... 

A simple diagnostic test has been devised for prostate cancer, using a specific molecular beacon mixed with the target DNA on a microscope slide. The DNA is treated to separate the strands and, provided there is the correct correspondence between the bases, in situ combination occurs between the bases on the molecular beacon and those on the strands. Thus, if fluorescence is observed, the DNA sample must contain the base sequence indicative of prostate cancer. 

Brazil M. High throughput screening—molecular beacons for DNA binding, Nature Reviews Drug Discovery 1 98-99 (2002). 

Dodge, A., Turcatti, G., Lawrence, I., de Rooji, N.R, Verpoorte, E., A Microfluidic platform using Molecular beacon-based temperature calibration for thermal dehybridization of surface-bound DNA. Anal Chem. 2004, 76, 1778-1787. 

Fig. 14. Examples of homogeneous hybridization assay methods (F luminophore, Q quencher, D donor, A acceptor). Thick lines represent DNA strands. Open circles on DNA strands indicate a SNP/mutation site for Molecular Beacon and insertion/deletion sites for dual FRET probe and dual FRET Molecular Beacon when these methods are applied to SNP/mutation typing or deletion/insertion detection. The solid circle on die strand indicates the complementary site.

Tsourkas et al. (2003) reported dual FRET molecular beacon assays, where the donor probe was labeled with either Eu3+ or Tb3+ complex of DTPA-csl24-ethylenediamine (and no quenchers attached). For the Eu3+ complex, the acceptor probe was Cy5-labeled (and no quenchers attached) and for the Tb complex, the acceptor probe was labeled with Cy3 or ROX as a fluorophore and with dabcyl as a quencher. They demonstrated that these pairs of probes detected DNA targets ( 50-mer) with high S/N. 

In molecular beacons fluorescence dequenching serves for detection of specific DNA segments in homogeneous solution, for example in real-time... 

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. 

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... 

Stoermer R, Cederquist KB, McFarland SK, Sha MY, Penn SG, 44. Keating CD. Coupling molecular beacons to barcoded metal nanowires for multiplexed, sealed chamber DNA bioassays. J. 

Kuhn H, Demidov W, Coull IM, Eiandaca MI, Gildea BD, Frank-Kamenetskii MD. Hybridization of DNA and PNA molecular beacons to single-stranded and double-stranded DNA targets. 42. J. Am. Chem. Soc. 2002 124 1097-1103. 

DNA- binding aptamers have been designed to bind to mRNA, g-quad-ruplexes, Tenascin-C, a protein found in the tumor matrix, and to thrombin. Aptamers have been selected as inhibitors of HIV-1 integrase, human RNase Hl, human pro-urokinase " and for the design of molecular beacons. An allosteric aptamer has been designed for binding as a colorimetric probe for cocaine. ... 

Molecular beacons (MBs) are hairpin-shaped oligonucleotides that report the presence of specific nucleic acids. The MBs have been immobihzed by Tan and co-workers [27] onto ultrasmall optical fibre probes through avidin-biotin binding. The MB-DNA biosensor detected its target DNA molecules, in real time, with selectivity for a single base-pair mismatch. This MB-DNA-biosensor was used by Perlette and Tan [28] for real-time monitoring of mRNA-DNA hybridization inside a living cell. 

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