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Seeing the light with PCR Gel electrophoresis remains a timeconsuming obstacle in the quest to develop speedy DNA analysis techniques. Even running a relatively quick mini-gel takes an eternity in the modern, high-throughput world. But Rudolf Rigler and colleagues at the Karolinska Institute (Sweden) have come up with an accelerated alternative. Using fluorescence correlation spectroscopy (FCS), they can detect PCR products in a matter of seconds. In FCS, labeled particles move through a small volume and are excited by a laser, and fluctuations in the fluorescent signal are measured and correlated. The amplitude of the resulting autocorrelation function changes with the particle concentration over time, providing a means to measure the amount of sample. An estimate of the particle's size is based on the diffusion time—longer molecules have larger diffusion constants and move more slowly. In the new technique, the researchers added tetramethylrhodamine-4-dUTP to a PCR reactor. The fluorescent label was in-
DNA weight watching Forget the Lean Cuisine. Yoshio Okahata and colleagues at the Tokyo Institute of Technology Gapan) want to see their DNA samples gain weight, not lose it. So the researchers are putting their quartzcrystal microbalances (QCMs) to work— watching for telltale mass increases as DNA polymerase does its duty. Most researchers have studied DNA elongation and the activity of polymerase using rapid-quenching gel electrophoresis or time-resolved fluorescence techniques, but Okahata and colleagues scrapped that approach in favor of the simpler in situ monitoring that a QCM could offer. The scientists immobilized oligonucleotides having a template and a primer on the electrode of a 27-MHz QCM using a biotin-avidin technique. Because these oligos covered only ~15% of the surface, there was enough room
corporated into the product, allowing detection but not interference with subsequent reactions such as product cleavage by restriction enzymes or digestion by an exonuclease. Incorporation into DNA quenched the fluorophore, reducing its quantum yield to less than a third of its free value, but the signal remained strong enough to allow the use of low levels of tetramethylrhodamine4-dUTP. Calibration curve showing relative translational diffusion By calculating the expected times of DNA fragments using FCS. The solid line (—) diffusion times for DNA fragshows the calculated values; the unconnected dots (•) show the measured values. ments of varying size, the researchers generated a calibration curve for determining molecular weight scientists note that such nonspecific priming based on the FCS diffusion time. The preis known to occur in other PCR amplificadicted results were within 2% of the experitions. They suggest that their technique is mental findings, and the sensitivity of the sensitive enough to detect products that nornew technique was several orders of magnimally cannot be seen with gel electrophoretude higher than that of gel electrophoresis. sis. In addition to such sensitivity, the speed Although the researchers detected formaand simplicity of the technique make it a tion of nonspecific "background" products at powerful, efficient method for detecting and low template concentrations (102 to 104 CODidentifying PCR products. {biochemistry ies)—even in the absence of template—the 1998,37112971-78) for the bulky Klenow fragment of DNA polymerase I (from E. colt) to bind. The researchers monitored the QCMsfrequencychanges over time and observed a mass increase corresponding to elongation and, later, a mass decrease indicating release of the enzyme from the completed doublestranded fragment When a second template, three times as long as the original was used instead, the mass increase was three times as great The researchers also determined the elongation rate, and the kinetic values were comparable with those determined by conventional methods Thus the QCM method seems to be as reliable as current techniques vet
cally and quantitatively using a single device. (J. Am. Chetn. Soc. 1998,120,8537-38)
simpler and more powerfijl Tt prn-
Experimental setup of a polymerase reaction using a vides the ability to detect each step template-primer oligonucleotide immobilized on a of the polymerase reaction kineti- 27-MHz quartz-rrv
