Molecular Beacons With and Without Quenchers

Modified nucleic acids have a wide range of applications in many areas of biochemistry. In particular, fluorescence-based nucleic acid systems have been studied extensively for their implementation in molecular biology as platforms for disease diagnosis. A hybridization probe is a fluorescent oligonucleotide used in DNA analysis, operating through sequence-specific complementary binding of a short synthetic oligonucleotide containing a fluorescent tag. Such fluorescent oligonucleotides play important roles in single-nucleotide polymorphism (SNP) typing, allowing both quantitative and qualitative analyses. Among them, stem–loop oligonucleotide probes have been developed to improve the specificity and selectivity toward target DNA. A molecular beacon (MB), a representative oligonucleotide probe having a stem–loop structure containing fluorescent and quencher units, is a probe used in biomolecular recognition. MB-based assays are fast, simple, and inexpensive and they enable real-time monitoring of nucleic acid responses in vivo and in vitro. Modifications of the structures and functions of MBs can lead to improved performance. For example, quencher-free molecular beacons (QF-MBs) are MBs, in which the quenching agent has been removed. Despite the absence of a quencher, QF-MBs can also identify specific target DNAs with high selectivity and sensitivity. MB and QF-MB probes have been applied widely in various fields, including SNP typing, monitoring of polymerase chain reactions (PCR), real-time detection of DNA–RNA hybridization in living cells, DNA mutation analysis, and disease diagnosis, including point-of-care (POC) testing.