B. H. Kim, Y. J. Seo and N. Venkatesan at Pohang University of Science and Technology in Korea explain why quencher-free molecular beacons mean a brighter outlook for medicine

Genetic analysis underpins DNA diagnostics. Swapping just a couple of letters in an individual's genome can cause an incurable genetic disorder, so identifying genetic differences is very important to diagnose and possibly cure certain diseases.

Unravelling the 'secret of life' by completing sequencing of the human genome, has led to our better understanding of genetic differences, be it between individuals, between parents and their offspring or normal and abnormal genes in genetic disorders. Such differences are expressed in terms of single nucleotide polymorphisms (SNPs, a single base change in a DNA sequence) or copy number variations (CNVs, when the number of copies of a gene varies). 

Initially, DNA analysis relied mainly on radiolabelled nucleotides. Now fluorescent techniques are being used increasingly. Analytical methods that use fluorescent probes to bind to particular DNA regions are now well-known. So-called molecular beacons (MBs), these probes are now being used during DNA amplification by polymerase chain reactions (PCR), to qualitatively as well as quantitatively estimate single or multiple gene sequences simultaneously. Similarly, different types of MBs are used in related applications such as protein analysis and to study protein-DNA interactions. In addition, MBs' suitability for probing the levels and kinetics of DNA photodamage, and as vehicles for photoinduced drug release has also been explored.

The conventional MB consists of a stable hairpin (stem-loop) oligonucleotide carrying a fluorophore at one end and a quencher at the other. In the hairpin form the fluorophore transfers its energy to the nearby quencher. When the target DNA is added, the hairpin opens out to bind to the DNA and the fluorophore is no longer quenched. So the target DNA is detected by an increase in fluorescence. This type of MB has been modified by a number of research groups to improve its detection limit, reduce its signal to noise ratio and to improve its stability against enzymes.

Quencher-free molecular beacons change fluorescence intensity as they bind to target DNA

Quencher-free molecular beacons (QF-MBs) are a recent addition to the family of modified MBs. These hairpin-shaped fluorescent oligonucleotides contain one or multiple fluorophores and can be broadly classified into two different types: mono-labelled, containing a fluorophore at the middle or one end of the oligonucleotide, and dual-labelled, containing two fluorophores at the stem or ends. In almost all, the entire or part of the hairpin's loop is complementary to the target oligonucleotide. 

None of these MBs has an additional quencher, but despite this each recognises fully complementary DNA. Quenching by photoelectron transfer between a nucleobase (usually guanine) and the fluorophore and changes in the fluorophore's microenvironment are major factors behind the successful working of the mono-labelled QF-MBs. Thus when QF-MB hybridises with its target DNA this brings about a change in one or both of these factors, resulting in a fluorescence intensity change. 

Mono-labelled QF-MBs can be used to reveal the nucleotide at an SNP and discriminate a fully matched target DNA from mismatched ones. The QF-MBs have notable advantages over the quencher-fluorophore MB systems. Nucleobases act as inbuilt quenchers, mono-labelled QF-MBs can be immobilised on a solid surface through the free end and, compared to the conventional MBs, preparation of mono-labelled QF-MBs is relatively easy and more economical. QF-MBs can be used in homogeneous as well as heterogeneous assay formats. Also, with one free end, the mono-labelled QF-MBs can be used as primers in PCR, to quantify target DNA levels with high sensitivity.

Researchers around the world continue to modify MBs to suit specific needs. Of these, QF-MBs are playing a significant role and, in the near future, should become one of the mainstream DNA analytical tools. Such technological advances will be the key to new diagnostic and treatment options. 

Read Kim  et al's tutorial review 'Quencher-free molecular beacons: a new strategy in fluorescence based nucleic acid analysis' in a forthcoming issue of Chemical Society Reviews.