A Non-PCR DNA Detection Method
Polymerase Chain Reaction (PCR), one of the broadest technologies to have emerged from biology in the last quarter century, has become so commonplace across fields from archeology to criminal investigations that it is hard to imagine what we ever did without it.
And harder, still, to imagine why we would want to do without it today.
Yet despite its broad application and its continued technological improvements, PCR requires a trained technician, necessitating either the expense of employing such a person in-house or the time and expense of sending samples to a dedicated facility.
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 inhibitorDNAenzyme 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 strandstraightening out the DNAand 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.
To read the article, "DNA Detection and Signal Amplification via an Engineered Allosteric Enzyme" by Alan Saghatelian, Kevin M. Guckian, Desiree A. Thayer, and M. Reza Ghadiri (J. Am. Chem. Soc., 125 (2), 344 -345, 2003), please see:
TSRI scientists have designed a new method to rapidly and sensitively detect specific sequences of DNA.