Molecular beacon: a multitask probe

... The specificity of hybridization of complementary sequences in DNA is the basic strategy for identifying target genes. For this, stem loop oligonucleotide probes have been developed in order to enhance the specificity and selectivity to the target DNA. Among stem loop oligonucleotides, molecular beacons are the recent probes used for biomolecular recognition reactions. Molecular beacon‐based assays are fast, simple, inexpensive, and enable real‐time monitoring of nucleic acid reactions both, in vivo and in vitro. This review has been designed to provide a better understanding of the different aspects of molecular beacons, e.g. structure, designing and applications in real‐time monitoring of nucleic acid amplification, detection of pathogens, nucleic acid–protein interaction, genetic analysis and array technology. Hybridization between complementary sequences forms the base of recent molecular approaches for the identification and detection of a particular gene. These hybridization techniques use specific labelled probes of small ssRNA or DNA fragments that can recognize and hybridize complementary sequence in the target RNA or DNA. Traditionally, there are different methods of radioactive/nonradioactive and fluorescent probe labelling. The hybridization steps constitute the labelling of probes, immobilization of target molecule on solid surface, hybridization to labelled probes, removal of un‐hybridized probes and finally the detection of bound probes. Removal of unbound probes and further washing steps usually disturb the equilibrium of nucleic acid hybridization event. Besides, due to the toxic effects of radioactive chemicals, the radiolabelled probes cannot be used to monitor real‐time amplification of DNA during PCR and for in vivo DNA synthesis. Because of these difficulties encountered with conventional probe labelling methods, research has been diverted towards oligonucleotide probe designing that enable dynamic, real‐time detection of nucleic acid amplification both in vivo and in vitro (Tyagi and Kramer 1996; Kostrikis et al. 1998; Tyagi et al. 1998). Molecular beacons were first developed by Tyagi and Kramer (1996) at the Public Health Research Institute, New York, USA. These are the fluorescent probes that produce fluorescence on hybridization with the complementary target. Molecular beacon probes can be used to monitor real‐time amplification during PCR, genetic analysis, detection of pathogens, gene mutation and in various other biological contexts. Research is still in progress for the modification of conventional molecular beacon probe to enhance the specificity and sensitivity. Scorpion probe is one of the variant of such modification that functions simultaneously as a PCR primer and a beacon probe (Whitcombe et al. 1999). Catalytic molecular beacons (Stojanovic et al. 2001) are another variant that can detect the target sequences without amplification. Another modification includes the construction of PNA‐DNA (Peptide Nucleic Acid) loop with molecular beacon, stemless beacon (Kuhn et al. 2002). PNA molecular beacons were also reported to be superior than conventional molecular beacon probes because of their faster hybridization kinetics, high signal to background ratio and much better specificity (Xi et al. 2003).