Molecular beacons target specificity

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. 

Usnally a nncleic acid probe that emits a signal only when bound to a specific target. An example is a molecular beacon. See Stohr, K., Hafner, B., Nolle., O. et al.. Species-specific identification of mycobacterial 16S rRNA PCR amplicons nsing smart probes. Anal. Chem. 77, 7195-7203, 2005. There are other examples of smart probes inclnding proteins (Wunder, A., Tung, C.-H., Mtiller-Ladner, U., Weissleder, R., and Mahmood, U., In vivo imaging of protease activity in arthritis, a novel approach for monitoring treatment response, Arthritis Rheumatism 50, 2459-2465, 2004) and chiral compounds (Tsukube, H. and Shinoda, S., Lanthanide complexes as smart... 

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. 

For studies of hematological malignancies, the sequence-specific fluorescence probe-based systems provide the advantage of an important further test for identification of the sequence of interest by virtue of hybridization of fluo-rescently labeled internal probes to the amplified target sequence. The most commonly used sequence-specific chemistries include the exonuclease (TaqMan), the linear hybridization probe, and the hairpin-based (Molecular Beacon) systems. (See Chapter 37 for further information on nucleic acid techniques.)... 

This study describes the development of a real-time PCR assay employing molecular beacons for detection of Salmonella. This assay combined the speed and specificity of PCR with the specificity and high sensitivity of molecular beacons. A 122 bp region of the himA gene was used as the target (6). The real-time PCR assay is highly specific and a detection limit of one single cell was achieved. 

Target Specificity. The specificity of the beacons was evaluated. Real-time PCR assays were conducted with Salmonella and E. coli as the initial templates. Although a 122 bp PCR fragment was amplified from both the Escherichia coli and Salmonella templates, only the Salmonella template elicited very strong fluorescence. This result clearly demonstrates the ability of molecular beacons to discriminate between very similar sequences. (Figure 4). 

Figure 3. Concept of PDT molecular beacons, where the targeting backbone may be either opened up or (as in this case) cleaved in vivo by the specific target, making the photosensitizer active courtesy G. Zheng, U. Pennsylvania, USA)...

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