By Aaron J. Sender
Like so many scientific discoveries, Mathias Howell stumbled into the new DNA detection method he now calls iFRET by accident. “The discovery was pretty much serendipity,” says Howell, 32, a PhD candidate at Karolinska Institute’s Center for Genomics and Bioinformatics in Stockholm.
He was working on tweaking a SNP detection assay he had developed in 1999 called DASH, or Dynamic Allele Specific Hybridization. “The recurring criticism was that DASH was not spectrally multiplexable,” says Howell. The assay used SYBR Green I dye, which is inexpensive and produces lots of fluorescence, but is non-specific: it glows when attached to any double-stranded DNA. “So we could only look at one SNP at a time. And all these other methods were able to see more than one SNP at a time,” says Howell.
To give it some more multiplexing punch, Howell began toying with adding a half-century-old detection method called FRET to his platform. FRET consists of two covalently linked dyes with similar emission/absorption spectra. When they are brought close together, for example when complimentary DNA strands hybridize, the donor molecule on one strand transfers energy to the acceptor molecule on the other. Different dyes produce different wavelength emissions, so FRET can easily be multiplexed.
The experiment had some strange results. “One of the controls had a standard reaction that I knew should work: I had immobilized target and the complimentary probe,” says Howell. “But there simply wasn’t any fluorescence. I couldn’t see anything. It was driving me crazy. I was thinking, ‘Was it dissipated as heat? Or was it a bad probe?’”
Howell spent a good part of a week trying to track down what happened. He had run this control hundreds of times in the past. Why was the SYBR Green not glowing? It turned out that while running the FRET and SYBR Green experiments in parallel, he had accidentally placed a target labeled for a FRET assay in the spot. “I was working late at night over the weekend trying to get a paper together,” explains Howell. “I was looking for the SYBR Green signal and it wasn’t there.” But looking at the full visible spectrum, he found that SYBR Green was acting as a very effective donor molecule.
By inadvertently merging FRET with SYBR Green, Howell had uncovered a new fluorescence signaling method with several advantages over either one of the other strategies. IFRET’s signal strength is twice as strong as SYBR Green and 40 times stronger than FRET.
Howell’s combo strategy is also much cleaner. “By using SYBR Green you get rid of that donor background that bleeds into your acceptor fluorescence.”
And by using SYBR Green as a donor molecule, iFret can take advantage of the same multiplexing capabilities that FRET allows without the expense of dual-labeled probes.
Howell and his advisor Anthony Brookes have started a company called DynaMetrix, which, using the DASH platform and iFret signaling, has built a prototype that can score SNPs at less than five cents apiece, according to Howell. They are now looking for a partner to develop a commercial-quality, automated instrument.
“People have run SYBR Green assays and FRET assays on the same machine before,” says Howell. “But nobody has ever tried to use SYBR Green as a donor before or even thought about it. It’s just not intuitive to do this. But now it seems so obvious. It’s like, aha, of course,” he says. “I love that kind of science.”