Molecular beacon

In molecular biology, a set of two hydrogen-bonded nucleotides on opposite complementary nucleic acid strands is called a base pair. In the classical Watson-Crick base pairing in DNA, adenine (A) always forms a base pair with thymine (T) and guanine (G) always forms a base pair with cytosine (C). In RNA, thymine is replaced by uracil (U). 

Cancer cells develop from alterations in genes (composed of nucleic acids), which confer growth advantage and the ability of the cancer to spread to different parts of the body. A novel way of achieving early detection of cancer is to detect nucleotide sequences of cancer-causing genes in living cells. 

Molecular beacons, developed in the mid-1990s, can be delivered into cells with high efficiency, where they are able to detect particular sequences of nucleotides that are indicative of certain types of cancers, thus aiding the early diagnosis of the disease. 

The molecular beacon essentially consists of four parts  

Molecular beacon unfolds and binds to the target nucleotide sequence 

Several proprietary fluorescence dye-based detection systems that characterize and quantify probe-bound nucleotide sequences have been developed and commercialized in recent years. For example, general and nonspecific DNA dyes can be used that bind with any double-stranded DNA (i.e., the probe-strand complex or otherwise) and are useful in gel electrophoresis. Much more sophisticated systems rely on oligonucleotide probes that incorporate fluorescent dyes that illuminate when a match between complementary strands are made. Included among the latter are TaqMan , molecular beacons, and Scorpion probes. 

Another type of probe that utilizes a signal system of fluorescent and quenching dyes is the molecular beacon (Fig. 16.6). Molecular beacons form a stem-loop structure when free in solution. The close proximity of the fluorochrome and quenching components prevents the probe 

Short-wavelength fluorophore and some nucleotides removed 

---

The three main categories of hybridization probes for real-time PCR are (1) cleavage based assays such as TaqMan, (2) displaceable probe assays such as Molecular Beacons and (3) probes which are incorporated directly into primers such as Scorpions. 

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. 

Fortin, N. Y. Mulchandani, A. Chen, W. Use of real-time polymerase chain reaction and molecular beacons for the detection of Escherichia coli 0157 H7. Anal. Biochem. 2001, 289, 281-288. 

Tyagi S, Kramer FR (1996) Molecular beacons probes that fluoresce upon hybridization. Nat Biotechnol 14 303-308... 

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... 

Bratu, D.P. (2006) Molecular beacons Fluorescent probes for detection of endogenous mRNAs in living cells. Meth. Mol. Biol. 319, 1-14. 

Tyagi, S. Kramer, F. R., Molecular beacons Probes that fluoresce upon hybridization, Nature Biotechnol. 1996, 14, 303 308... 

Molecular beacons are single-stranded hairpin-shaped nucleotide probes. In the presence of the target nucleotide sequence the molecular beacon unfolds, binds and fluoresces (Figure 6.13). 

Loop this is the nucleotide region of the molecular beacon, which is complementary to the target nucleotide sequence. 

Figure 6.13 Mode of action of a molecular beacon. Fluorescence is only observed when the beacon and the target nucleotide bind together...

A wavelength-shifting molecular beacon contains three labels ... 

Figure 6.14 Principle of operation of a wavelength-shifting molecular beacon...

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). 

These include Molecular Beacons (Fig. 6A) coupled with NASBA or Sunrise coupled with PCR (Fig. 6B). The Molecular Beacons patent bears striking similarities to the Sunrise patents. 

ML Marras, S. A., Kramer, F. R., et al. Multiplex detection of single-nucleotide variations using molecular beacons. Genet. Anal. 14(5—6), 151—156 (1999). 

T4. Tyagi, S., Bratu, D. R, etal. Multicolor molecular beacons for allele discrimination. Wat. Biotech-nol 16(1), 49-53 (1998). 

---