Capillary Electrophoresis, Meet Lab-on-a-Chip; Virtual NMR; Researchers Map Worm ORFeome

TECH BRIEF | Researchers Map Worm ORFeome Courtesy of Marc Vidal Citing the need to verify genome annotations and to provide a resource for functional genomics, Marc Vidal of the Dana-Farber Cancer Institute and an international team of researchers have tackled what they call the "ORFeome," the complete set of protein-encoding open-reading frames (ORFs) in the Caenorhabditis elegans genome (Nat Genet, advance online publication, DOI:10.1038/ng1140, April 7, 2003). Using annotation data, the team designed 19,477 primer pairs to precisely amplify each worm coding sequence (excluding untranslated segments), attempted amplification from a highly representative cDNA library, and cloned the resulting 11,984 ORFs into Invitrogen's Gateway system to make ORFeome 1.1. The team used this collection to verify 4,365 predicted but experimentally unconfirmed ORFs; assess the prevalence of alternative splicing variants and the accuracy of intron-exon boundary predictions (nearly one-third were incorrect); and demonstrate its utility in "interactome" mapping and in proteomics research. The ORFeome, says Vidal, differs from a cDNA library in several respects. First, ORFeome clones are always full-length, stored individually in 96-well plates, and present exactly once, with rare and abundant transcripts represented equally. Second, thanks to Gateway technology--a recombinase-based system--these clones can easily be shuffled from vector to vector as the need arises. Finally, because clones contain no noncoding sequences, researchers can make gene fusions without fretting over frame shifts. ORFeome research rolls on, says Vidal, with version 2.1 and 3.1 in development and 4.1 under consideration. --Jeffrey M. Perkel SOFTWARE WATCH | Virtual NMR Courtesy of David Fushman Few laboratory techniques can rival the mystery underlying nuclear magnetic resonance (NMR) spectrometry. Computer simulators can help, but, says David Fushman, associate professor of chemistry and biochemistry, University of Maryland, these often require sophisticated programming skills or assume ideal experimental conditions. Fushman's Virtual NMR Spectrometer (www.vsnmr.org), a freeware Matlab application, circumvents these limitations. Users can specify and probe up to 12 interacting nuclei (1H, 13C, or 15N) with either "canned" or novel pulse sequences, tweaking parameters, delays, and pulse shapes to see how they affect results. The program supports n-dimensional experiments, pulsed-field gradients, and nonideal conditions. The pulse sequence translator recognizes programs written for Bruker instruments, but a Varian interpreter is under development. Sami Heikkinen, research scientist, Helsinki University Central Hospital, employs the software for experimental planning. The simulated spectra, he says, "were in good agreement with the measured ones." Olivier Walker, assistant professor, University of Nancy, France, uses the software as a teaching tool. "I highly recommend it for all the people who want to discover NMR and more," he says. The Virtual NMR Spectrometer is currently in the prebeta testing phase; Fushman expects to release a full beta version in June. In development: code for chemical exchange, complex decoupling schemes, and arbitrary RF pulses; libraries of predefined spin systems and pulse sequences; extension to other nuclei; and new modules for data processing, including interfaces to commercial analysis packages. --Jeffrey M. Perkel PATENT WATCH | Capillary Electrophoresis, Meet Lab-on-a-Chip According to Shaorong Liu, an associate professor of chemistry and biochemistry at Texas Tech University, current high-throughput DNA sequencing separation via "labs-on-a-chip" involves tedious processes that are poorly integrated and unnecessarily expensive. "An unresolved need exists," Liu writes in his recently awarded US patent "... for the development of high speed, high-throughput DNA sequencing methods and apparatus that are capable of reading DNA sequences significantly longer than 500 bases, using small amounts of DNA sample in a small sample volume" (S. Liu, "Microfabricated injector and capillary array assembly for high-resolution and high throughput separation," US patent 6,533,914, issued March 18, 2003). Liu's solution: a hybrid device consisting of a microfabricated chip injector attached to an array of one or more capillaries with zero dead-volume. He told The Scientist that the apparatus "enables the integration of these various microchips and capillaries for high-throughput and high-quality DNA sequencing." From the perspective of capillary-based sequencing, says Liu, the invention "allows injection of narrow sample bands so as to increase the separation efficiency, and permits additional sample purification so as to improve the system robustness," as well as enabling integration of sample preparation with sequencing preparation. From the point-of-view of microchip-based sequencing, the invention "provides an effective means to extend the straight separation channels on a space-limited microchip," he says. MicroChem Solutions, of Lubbock, Texas, has a nonexclusive right-of-use to the patent, Liu says, but it is available for licensing by other companies. --Ivan Oransky function sendData() { document.frm.pathName.value = location.pathname; result = false if (document.frm.score[0].checked) result = true; if (document.frm.score[1].checked) result = true; if (document.frm.score[2].checked) result = true; if (document.frm.score[3].checked) result = true; if (document.frm.score[4].checked) result = true; if (!result) alert("Please select") return result; } .myradio{background-color : #94bee5} Please indicate on a 1 - 5 scale how strongly you would recommend this article to your colleagues? Not recommended 1 2 3 4 5 Highly recommended Please register your vote

Capillary Electrophoresis, Meet Lab-on-a-Chip; Virtual NMR; Researchers Map Worm ORFeome

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