Researchers at The Scripts Research Institute have developed a new method for detecting specific DNA sequences.
Now TSRI Ph.D. graduate and current research associate Alan Saghatelian, TSRI graduate student Desiree Thayer, research associate Kevin Guckian, and Professor Reza Ghadiri in the Department of Chemistry have designed a non-PCR method for detecting specific sequences of nucleic acid that may have advantages over PCR, especially in such situations as field work and point-of-care medicine where the technology could be used by non-specialists. The new method is exquisitely sensitive and quite fast, according to Ghadiri, detecting as minute a sample as 10 femtomoles of DNA in less than three minutes. The method makes use of a detection system based on an inhibitor–DNA–enzyme complex. Specifically, the complex is composed of an enzyme, a single-stranded piece of DNA covalently attached to the enzyme, and, at the end of this DNA strand, an "intramolecular" inhibitor. The complex is able to "detect" pieces of DNA that are complimentary to its single strand of DNA. When complimentary DNA is not present, the single strand of DNA in the complex is flexible enough that it can loop around, allowing the inhibitor to occupy the binding site of the enzyme. But when complimentary DNA is present, the complimentary DNA forms a duplex with the complex's single strand—straightening out the DNA—and the inhibitor at the end on this duplex can no longer occupy the enzyme's binding site, enabling the enzyme to cleave its substrate. Ghadiri and his colleagues selected a fluorophoric substrate so that this cleavage releases energy in the form of easily detected fluorescence, signaling the presence of complimentary DNA. The sensitivity of the method comes from the fact that the system is self-amplifying. Any one molecule of DNA that hybridizes to one complex turns on that one enzyme, which can then do multiple turnovers of the substrate.
This is not a general sequencing method. Its designed to detect specific sequences of DNA. Its advantage over the existing polymerase chain reaction method is the potential ability to build devices that use it that do not require a trained technician. This will lower costs and allow use in a larger range of settings.
Its not clear from the press release what this technique's limitations might be. Could a partially matching fragment cause the method to report a match? Is it sensitive down to the level of a single nucleotide polymorphism (SNP) difference? It would be a lot more useful medically if it was. Eventually specific SNPs will be linked to medically useful factors such as drug sensitivities and incompatibilities. At that point what would be needed is the ability of a doctor to test for a specific SNP in order to choose the best drug treatment.
|Share |||Randall Parker, 2003 January 19 04:21 PM Biotech Assay Tools|