Researchers in the US have prepared a molecular beacon that allows the quantitative detection of DNA.

Molecular beacons are able to detect specific DNA or RNA sequences in living cells. Nicholas Turro from Columbia University and colleagues have modified the basic beacon design to incorporate fluorescence resonance energy transfer (Fret). 

The basic beacon design consists of a single strand of nucleic acid with a fluorescent group and a quencher group attached at opposite ends. Normally the beacon resembles a hairpin, a 'stem-loop' structure. In this form the fluorescent and quencher groups are held together and no fluorescence is emitted. 

The sequence of the loop section is complementary to the target DNA or RNA sequence. When the target sequence is added, the beacon shifts to a stem-open form to better bind the target. The fluorescent and quencher groups become separated and the fluorescent signal can be detected.

Turro's Fret beacon replaces the fluorescent and quencher groups with two fluorescent groups, a donor and an acceptor. In the stem-closed form the acceptor absorbs fluorescence from the donor by Fret and only emission from the acceptor is seen. In the stem-open form, with the groups held apart, very little Fret can occur and the fluorescence of the donor dominates. Since the donor and acceptor emit at different wavelengths, the change in beacon shape is observed as a change in the wavelength of the emitted light.

The modified beacon prevents 'false positive' signals which have been associated with classical molecular beacons due to incomplete quenching, said Turro. The Fret beacon also allows improved quantitative analysis of the target sequences, by measuring the ratio of the fluorescent signatures. 'Ratiometric analysis is generally superior compared to pure intensity changes,' said Turro. 

Michael J Spencelayh