Molecular beacon

In order for molecular beacon assays to work correctly, molecular beacons must be of the highest quality.

At Biolegio we have studied different methods to make molecular beacons. These studies resulted in the production of high quality molecular beacons. The purity of every molecular beacon preperation is checked and only the purest fraction will be selected for your final product.
The Molecular Beacons sold by Biolegio will have a minimum signal-to-background ratio of at least 25 to 1 when tested against the complementary target sequence.

Overview

In 1996 Tyagi and Kramer described novel hairpin-shaped nucleic acid detection probes, called "molecular beacons", that undergo a conformational transition when they bind to their target that enables them to fluoresce brightly (1).

The loop portion of a molecular beacon is a probe sequence that is complementary to a predetermined target sequence and the stem is formed by the annealing of complementary arm sequences that are present on either side of the probe sequence. A fluorophore is covalently attached to the end of one arm and a nonfluorescent quencher is covalently attached to the end of the other arm. In the absence of target, the stem keeps the fluorophore and the quencher in close proximity to each other, preventing fluorescence. However, when molecular beacons bind to their target they undergo a conformational change that restores the fluorescence of the internally quenched fluorophore.

Figure 1. Interaction of a molecular beacon with its target.
Figure 1. Interaction of a molecular beacon with its target.

Multicolor molecular beacons for multiplex assays.
The hairpin structure of a molecular beacon enables the use of a wide variety of differently colored fluorophores (Tyagi et al., 1998). In the hairpin conformation, the fluorophore and quencher are so close to each other, that direct transfer of energy is possible. Therefore the quencher, the non-fluorescent chromophore DABCYL, can quench any fluorophore (Fig.2). This quenching is independent of the overlap between the emission spectrum of the fluorophore with the absorption spectrum of the quencher.


A solution of each molecular beacon was placed in a pair of test tubes. The molecular beacons contained (left to right) coumarin (blue), EDANS (blue-green), fluorescein (green), Lucifer yellow, tetramethylrhodamine (orange), and Texas red. All molecular beacons contain DABCYL as a quencher. Complementary single-stranded oligonucleotides were added to the left tube of each pair, and the tubes were illuminated with a broad-wavelength ultraviolet lamp. (Tyagi et al., 1998).  

Fluorogenic response of differently colored molecular beacons to the addition of target.
Figure 2. Fluorogenic response of differently colored molecular beacons to the addition of target.

The extraordinary specificity of molecular beacons
The hairpin stem of molecular beacons also enhances specificity. Molecular beacons readily distinguish targets that differ by only a single nucleotide, and they are significantly more specific than conventional oligonucleotide probes of equivalent length (Tyagi et al., 1998, Marras et al., 1999, Täpp et al., 2000). The enhanced specificity is due to their ability to form a stem-and-loop structure. The specificity of molecular beacons can be modulated by altering the degree of constraint placed on their conformation (Bonnet et al., 1999). An example of a multiplex molecular beacon assay in which in one tube both the wildtype and mutant allele are analyzed is shown in figure 3 (The principle of spectral genotyping. (Kostrikis et al., 1998)). 		 The extraordinary specificity of molecular beacons

In summary, the hairpin stem of a molecular beacon enhances specificity, enabling accurate detection of single-nucleotide differences. Furthermore, the hairpin stem brings the fluorophore so close to the quencher that an entire palette of colored fluorophores can be used. Thus, mixtures of multicolored molecular beacons can be formulated that identify closely related allelic variants of a target by the color of the fluorescent signal.

Molecular beacons have been successfully applied in different molecular diagnostic studies (Giesendorf et al., 1998; Kostrikis et al., 1998; Piatek et al., 1998), for example, multicolor molecular beacons were used to develop an extremely sensitive, high-throuhput clinical assay that simultaneously detects four different retroviruses (Vet et al., 1999). Moreover, molecular beacons have been used to directly detect specific messenger RNAs in living cells (Matsuo et al., 1998; Sokol et al., 1998).

Available Fluorophores

Molecular Beacons are synthesized with a quencher at the 3’-site and a fluorophore at the 5’-site. The most frequently used quencher at this moment is Dabcyl, however, there are other quenchers available, please inquire. As a fluorophore you can choose between different dyes. The most commonly used are FAM, HEX, TET, TAMRA, Cy3 and Cy5. If you would like another kind of label as fluorphore, please contact Biolegio for the possibilities.
For more information see the section quenchers,  Black Hole QuencherTM.

Biolegio Offers:

  • High Quality Molecular Beacons
  • Guaranteed signal-to-background ratio of at least 20 to 1
  • Free complementary target sequence delivered together with your Molecular Beacon
  • The possibiltiy to have a complete determination of the melting curves of your Molecular Beacons
  • Design of your Molecular Beacons by Biolegio

Relevant literature

  • Bonnet, G., Tyagi, S., Libchaber, A., Kramer, F.R. 1999. Thermodynamic basis of the enhanced specificity of structured DNA probes. Proc Natl Acad Sci USA 96:6171-6176.
  • Giesendorf, B.A.J., Vet, J.A.M., Tyagi S., Mensink, E.J.M.G., Trijbels, F.J.M., Blom H.J. 1998. Molecular beacons: a new approach for semi-automated mutation analysis. Clinical Chemistry 44:482-486
  • Kostrikis, L.G., Tyagi, S., Mhlangha, M.M., Ho, D.D., and Kramer F.R. 1998. Spectral Genotyping of human alleles. Science 279:1228-1229
  • Leone, G., van Schijndel, H., van Gemen, B., Kramer, F.R., and Schoen C.D. 1998 Molecular beacon probes combined with amplification by NASBA enable homogenous, real-time detection of RNA. Nucl Acid Res 26:2150-2155.
  • Marras, S.A.E., Kramer, F.R., Tyagi, S. 1999. Multiplex detection of single-nucleotide variation using molecular beacons. Genetic Analysis 14:151-156.
  • Matsuo, T. 1998. In situ visualization of messenger RNA for basic fibroblast growth factor in living cells. Biochim Biophys Acta 1379: 178-184.
  • Piatek, A., Tyagi, S., Pol, A.C., Telenti, A., Miller, L.P., Kramer, F.R., and Alland, D. 1998. Molecular beacon sequence analysis for detecting drug resistance in Mycobacterium tuberculosis. Nature Biotechnology 16:359-363.
  • Sokol, D.L., Zhang, X., Lu, P., Gewirtz, A.M. 1998. Real time detection of DNA.RNA hybridization in living cells. Proc Natl Acad Sci U S A 95:11538-11543.
  • Tyagi, S., and Kramer, F.R. 1996. Molecular beacons: probes that fluoresce upon hybridization. Nature Biotechnology 14:303-308.
  • Tyagi, S., Bratu, D.P., and Kramer, F.R. 1998. Multicolor molecular beacons for allele discrimination. Nature Biotechnology 16: 49-53.
  • Vet J.A., Majithia A.R., Marras S.A., Tyagi S., Dube S., Poiesz B.J.,Kramer F.R. 1999. Multiplex detection of four pathogenic retroviruses using molecular beacons. Proc Natl Acad Sci USA 96:6394-6399.
  • Täpp, I., Malmberg, L., Rennel, E., Wik, M., Syvänen. 2000. Homogenous scoring of single-nucleotide polymorphisms: comparison of the 5’-nuclease TaqMan assay and molecular beacon probes. Biotechniques 28: 732-738.

Biolegio is licensed by the Public Health Research Institute of New York to manufacture Molecular Beacons for research use. Order Now