Fluorescent dye for DNA detection

Scientists at the US Department of Energy’s (DOE) Brookhaven National Laboratory have developed a highly sensitive way to detect specific sequences of DNA. Writing in the journal Chemistry of Materials, the researchers explained that their “label-free, sequence specific DNA sensor [is] based on fluorescence resonant energy transfer (FRET) occurring between a cationic conjugated polyelectrolyte and a small intercalating dye, malachite green chloride”.

“The sensors we’ve developed use a light-absorbing polymer to amplify the fluorescent signal of a dye that emits light only when it binds between two matched pieces of DNA,” said Mircea Cotlet, a physical chemist at Brookhaven’s Center for Functional Nanomaterials and leader of the research.

The method has high potential to be made field deployable for rapid analysis of crime-scene evidence and to mount a more knowledgeable, speedy response to bioterror threats. It is rapid and requires no expensive equipment - just a conventional laboratory fluorimeter. According to first author Zhongwei Liu, “The dye we use is hundreds of times cheaper than popular commercial intercalating dyes.”

When DNA sequences are complementary, one fluorescent dye molecule (red rectangle) binds between each matching base pair. The conjugated polymer (blue) wraps around the DNA, absorbs light and transfers the energy (via fluorescence resonant energy transfer, or FRET) to the dye to amplify the photoluminescent (PL) signal (red arrow). When DNA strands don't match perfectly, fewer dye molecules bind and the magnitude of the PL signal is reduced.

Unbound, the green-coloured molecule absorbs red light but does not emit light. “But when it inserts itself in the grooves of the DNA, the dye becomes fluorescent,” said Liu. “And so far it is the only dye that can intercalate so densely with DNA - meaning exactly one dye molecule binds between each complementary base pair of the DNA double helix.”

That means the strength of the fluorescent signal is directly related to how many dye molecules are bound - and how closely an unknown DNA sample matches a probe strand used for testing. As soon as there’s a mismatch, even at just one ‘rung’ on the DNA ladder, a dye molecule won’t bind and the signal will weaken. Two mismatches results in a proportional drop in signal strength, and so on.

To amplify these signals, the scientists add a conjugated polymer. These light-absorbing materials are used for harvesting sunlight in solar cells, but Cotlet noted that the researchers could make them water soluble and compatible with biomolecules like DNA. Synthesised by Hsing-Lin Wang, a collaborator at DOE’s Los Alamos National Laboratory, the polymers were functionalised with side chains that carry a positive charge, allowing them to naturally bind with negatively charged DNA via electrostatic interactions.

“The polymer wraps and follows the helix of the DNA,” Cotlet said. “This configuration brings the polymer in close proximity with the DNA-bound dye molecules and also enhances the polymer’s ability to absorb and emit light. Both of these factors help with the transfer of energy to the dye-intercalated DNA and increase the sensitivity of the biosensor.”

So if scientists want to know whether two pieces of DNA are identical - say a known sequence from an anthrax spore and one from a suspicious-looking white powder - all they have to do is mix the samples, dye and polymer in a test tube, turn on the light and let the results shine for themselves.

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