Sequence specific

Detecting specific sequences of DNA quickly and cheaply is a prerequisite for developing widely usable gene sensors. Michael Gross reports on progress so far.

Imagine a new device, maybe the size of a pen, with a metal tip and a small display screen like a digital thermometer. You scrape the tip over the inside of your cheek and press a button, and within a second, it can tell you whether or not you carry the gene variant that will lead to side effects if you take a certain medication. An identical looking stick, equipped with different molecular probes on the inside, might tell you whether the drinking water in Bangladesh is free from cholera germs, or whether an escaped convict has been involved in a given crime. These would be just some of the numerous potential applications if one could build a small, simple, and reusable sensor for specific DNA sequences.

There are, of course, biochemical approaches to this task. You could fragment the DNA in question, amplify the sequence of interest by PCR with specific primers, and/or analyse it on gels. All these require equipment, reagents and a specialised laboratory. For the gene test that is one day to fit into a pen-sized tool that anybody could use anywhere in the world, all complicated chemical procedures are taboo. There is only one reaction of interest: the target DNA (from the sample investigated) forms a double helix with the complementary probe DNA and thereby triggers a signal, most likely to be electronic or optical, that will be passed on to appear as the required information on the display.

A first promising approach towards simple and sequence-specific DNA recognition with electronic read-out was made in the mid-1990s by Faiz Kayyem, who used two kinds of DNA molecules - the immobilised capture strand and the redox-labelled probe strand - to hybridise with the target strand to be detected. In this so-called sandwich approach, only the combination of three molecules allows the signal to be generated. Kayyem founded the company Clinical Microsensors (now part of Motorola), which is now nearing the commercialisation of a desktop instrument for clinical laboratories.

While the sandwich sensor requires the addition of a reagent solution, two newly developed DNA sensors achieve the goal of high-sensitivity reagentless detection. Both use a DNA stem-loop structure - immobilised on a solid surface with one end and labelled at the other - as the probe. If and when the target DNA binds to the probe sequence included in the loop, the buckled up molecule stretches out and the label is removed from the surface. This change is detected by fluorescence in one study, and by cyclovoltammetry in the other.

Todd Krauss and his group at the University of Rochester in New York transferred the ’molecular beacon’ approach, where the soluble biomolecule contains both a fluorescent label and a quencher, to the solid state. They designed their DNA probe such that the fluorescence label (rhodamin) was so close to the gold substrate that the fluorescence was quenched with over 95 per cent efficiency. On recognition of the matching one out of two target sequences, which were derived from naturally occurring genes that confer resistance to an antibiotic (methicillin), the rhodamin group was distanced from the surface, resulting in a more than 20-fold increase in fluorescence intensity. Thus far, the technique can detect DNA concentrations as low as 10nM, but the authors anticipate that they can push the sensitivity further by optimising the technology.

In an alternative approach based on the same principle, Kevin Plaxco and his group at the University of California at Santa Barbara used cyclovoltammetry to detect the unfolding of the DNA hairpin. They too attached one end of the probe DNA to a gold surface, but to the other end they attached a ferrocene label (see Fig). While the probe DNA is ’buckled up’ in a hairpin, the ferrocene is close to the gold surface and takes part in redox reactions that lead to the characteristic cyclovoltammetric profile, with peak currents at about 100nA. Hybridisation with the target DNA abolishes this signal.

The E-DNA sensor: an electrochemically labelled DNA-hairpin (left) straightens on recognising its target sequence (right) and thereby removes the ferrocene (Fc) label from the gold surface

To avoid any false positive signals that might result from degradation of the probe DNA or removal of the ferrocene by other means, the team has included a second DNA sequence as a negative control. This second DNA sequence can be tagged with a different electrochemical label, corresponding to the different colours of fluorescence labelling.

The current version of the device, which the Californian researchers have dubbed ’E-DNA sensor’ can selectively detect a target gene sequence at a concentration limit of 10pM (5fmol in 0.5ml solution), which is comparable to the sensitivity of the sandwich sensor. This performance from a robust and user-friendly device is a major improvement, but it is not quite sufficient to detect most pathogens without amplification by PCR. Like the Rochester team with the fluorescence-based sensor, Plaxco and his group are working to improve the sensitivity of their device.

While the pen-sized gizmo described above is in the realm of science fiction for now, it may not stay there for much longer.

Source: Chemistry in Britain

Acknowledgements

Michael Gross is a science writer in residence at the school of crystallography, Birkbeck College, University of London. He can be contacted via the  prose and passion web page.