Molecular Diagnostics

Molecular beacons

Introduction to beacon technology

[PICK Comment. This was an article written before the introduction of the current generation of Abbott tests]].

Molecular beacons were first described by Tyagi et al., 1998. Molecular beacons oligonucleotide probes have a compact hairpin structure, with a fluorophore attached to one end and a quencher molecule attached to the other end. In its native state, the fluorophore beacon is quenched by virtue of its proximity to the quencher molecule. Upon hybridization with its complementary oligonucleotide target, fluorescence is elicited due to a conformational change that results in separation of the fluorophore and quencher molecule [Ramachandran et al., 2003]. Molecular beacons technology is therefore potentially adaptable to any kind of assay involving interaction with an oligonucleotide probe. Furthermore the beacon fluorophore may be used to measure other interactions, such as between antigen and antibody [Wei & Herron, 2002] or for imaging processes within living cells [Wouter et al., 2001]. Molecular beacons may be used for in vivo imaging in conjunction with computer enhanced tomography [Ntziachristos et al., 2002]. To avoid labelling of the analyte, fluorophore labelled DNA hairpins acting as molecular beacons may be attached to gold films to function as biosensors [Du et al., 2003]. The performance of the molecular beacons themselves is characterized by the stability of their stem-and-loop structures and that of the probe-target duplex. This in turn depends on the free energies accompanying their formation. Thermodynamic models to describe this interaction have been described [Szemes & Schoen, 2003; Tsourkas et al., 2003b], leading to a design method for RNA detection by molecular beacons which is adaptable to microarrays [Szemes & Schoen, 2003]. Molecular beacons can differentiate between bound and unbound probes in homogeneous hybridization assays with a high signal-to-background ratio and enhanced specificity compared with linear oligonucleotides but need to be protected from degradation with endogenous endonucleases [Tsourkas et al., 2003a]. Their high specificity makes them particularly useful for detecting single nucleotide polymorphisms [Marras et al., 2003]. Simple algorithms and web tools have been described to design the sequences of molecular beacons [Monroe & Haselton, 2003]. A database of primers and probes for real time PCR, including molecular beacons, is available on the web [Pattyn et al., 2003] [PICK but suffers from irritating pop up advertising]. The database may be queried using the official gene name, a sequence, Locus link or SNP identifier. Researchers are encouraged to submit their validated primer and probe sequences.

Abbott Molecular Beacons test for C. trachomatis

A preliminary description of an Abbott molecular beacons PCR-based test [which is understood to be the replacement for the withdrawn LCx LCR test for genital C. trachomatis infection] has been published [Abravaya et al., 2003]. The authors point out that molecular beacons allow multiplex detection of PCR products in real time in a homogeneous assay format. The real time detection is inherently quantitative and affords a greater dynamic range than end-point detection methods. In the Abbott formulation, reactions in a homogeneous assay format are sealed before amplification takes place, providing improved contamination control. A single cycler/reader instrument is coupled with automated sample preparation ease of use and higher specimen throughput. A multiplex qualitative assay that detects C. trachomatis and Neisseria gonorrhoeae, together with an internal control, has been developed and is expected to be marketed [Abravaya et al., 2003].

As yet there are few published comparisons of molecular beacons technology with other nucleic acid-based diagnostic techniques on clinical material. However a comparison of Taqman and molecular beacons probes for the real time PCR of Epstein Barr Virus and of cytomegalovirus found that both TaqMan and molecular beacon assays had comparable performance, being linear between 10 - 107 viral genomes/reaction. Both approaches were considered to give accurate, rapid, and reliable assays of these viruses in patient material [Jebbink et al., 2003]. Other applications will readily be found in PubMed using the search term 'molecular beacons'.

MeWard 2003.

SEE ALSO: Research methods: Quantitative Pcr.

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References

Abravaya K, Huff J, Marshall R, Merchant B, Mullen C, Schneider G, Robinson J. (2003). Molecular beacons as diagnostic tools: technology and applications. Clinical Chemisty and Laboratory Medicine 41, 468 - 474.

Du, H., Disney, M. D., Miller, B. L. & Krauss, T. D. (2003). Hybridization-based unquenching of DNA hairpins on Au surfaces: prototypical "molecular beacon" biosensors. Journal of the American Chemical Society 125, 4012 - 4013.

Jebbink, J., Bai, X., Rogers, B. B., Dawson, D. B., Scheuermann, R. H. & Domiati-Saad, R. (2003). Development of Real-Time PCR Assays for the Quantitative Detection of Epstein-Barr Virus and Cytomegalovirus, Comparison of TaqMan Probes, and Molecular Beacons. Journal of Molecular Diagnostics 5, 15 - 20.

Marras, S. A., Kramer, F. R. & Tyagi, S. (2003). Genotyping SNPs with molecular beacons. Methods in Molecular Biology 212, 111 - 128.

Monroe, W. T. & Haselton, F. R. (2003). Molecular beacon sequence design algorithm. Biotechniques 34, 68 - 70 & 72 - 73.

Ntziachristos, V., Tung, C. H., Bremer, C. & Weissleder R. (2003). Fluorescence molecular tomography resolves protease activity in vivo. Nature Medicine 8, 757 - 760.

Pattyn, F., Speleman, F., De Paepe, A. & Vandesompele, J. (2003). RTPrimerDB: the real-time PCR primer and probe database. Nucleic Acids Research 31, 122 - 123. Web link to database

Ramachandran, A., Zhang, M., Goad, D., Olah, G., Malayer, J. R. & El Rassi, Z. (2003). Capillary electrophoresis and fluorescence studies on molecular beacon-based variable length oligonucleotide target discrimination. Electrophoresis 24, 70 - 77.

Szemes, M. & Schoen, C. D. (2003). Design of molecular beacons for AmpliDet RNA assay-Characterization of binding stability and probe specificity. Analytical Biochemistry 315, 189 - 201.

Tsourkas, A., Behlke, M. A. & Bao, G. (2003a). Hybridization of 2'-O-methyl and 2'-deoxy molecular beacons to RNA and DNA targets. Nucleic Acids Research 30, 5168 - 5174.

Tsourkas, A., Behlke, M. A., Rose, S. D. & Bao, G. (2003b). Hybridization kinetics and thermodynamics of molecular beacons. Nucleic Acids Research 31, 1319 - 1330.

Tyagi, S., Bratu, D. P. & Kramer, F. R. (1998). Multicolor molecular beacons for allele discrimination. Nature Biotechnology 16, 49 - 53.

Wei, A. P. & Herron, J. N. (2002). Bifluorophoric molecules as fluorescent beacons for antibody-antigen binding. Journal of Molecular Recognition 15, 311 - 320.

Wouters, F. S., Verveer, P. J. & Bastiaens, P. I. (2001). Imaging biochemistry inside cells. Trends in Cell Biology 11, 203 - 211.

SEE ALSO: Research methods: Quantitative PCR.

NEXT: SDA. The Becton Dickinson ProbeTec.

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Topic revision: r2 - 2011-03-12 - MeWard
 
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