Fast DNA sequencing technique developed
SAN FRANCISCO Chemists at the University of Wisconsin, Madison, have developed an approach for significantly increasing the rate at which the fragments produced in DNA sequencing reactions can be analyzed by gel electrophoresis. The research was described at a symposium sponsored by the Biotechnology Secretariat. The technique, which utilizes ultrathin gels, provides more than 9000 bases of DNA sequence data per hour. Current commercial sequencing instruments produce about 1350 bases of DNA sequence data per hour. And Lloyd M. Smith, an assistant chemistry professor at Wisconsin who developed the technique, says that fairly straightforward extensions of the current design will enable construction of a system with a throughput of 26,000 bases per hour. Smith points out that high-speed DNA sequencing is an absolute necessity if the human genome project and related sequencing efforts are to succeed. To date, about 50 million base pairs of DNA sequence have been published, about 6 million of which are from the human genome. The longest individual sequence that has been determined is about 300,000 base pairs in
Smith: method uses ultrathin gels
is not simply separating and detecting DNA fragments. 'That's only one part of the problem/' he says. 'There is also generating the fragments and handling the data that are generated. All of these are significant problems that remain to be overcome for the human genome project to proceed." Rudy Baum
length. The DNA in the human genome contains on the order of 3 billion base pairs, which, Smith says, represents "a big hurdle to jump in sequencing capability." Currently, virtually all DNA sequencing is based upon the separation of DNA fragments in high-resolution polyacrylamide gels of 200 to 400 |Lim thick. Over the past two years, Smith and others have shown that the time required for gel electrophoretic separations of DNA can be reduced using capillary gel electrophoresis. In capillary gel electrophresis, fluorescently labeled products of sequencing reactions are separated and detected in 50-|Lim-diameter polyacrylamide gels cast in fused silica capillaries. The Gold clusters embedded in a fluorocarhigh efficiency of heat dissipation in bon polymer film, when illuminated these capillaries permits much larger with a focused laser beam, can be electric fields to be applied without made to melt and coalesce into a condamage from heating, yielding a con- ducting metal line. This new approach comitant increase in the speed of the can produce microcircuit patterns dielectrophoretic separations, Smith says. rectly on a fluoropolymer carrier, says Capillary gel electrophoresis is, how- Paul B. Comita, a physical chemist who ever, inherently a serial technique so led the research at IBM Almaden Rethat, although the separations are faster, search Center in San Jose, Calif. overall throughput of DNA sequence by Comita's work is "very novel and inthis method is roughly comparable to teresting and has fundamental and practhat of existing instruments. One could, tical applications/' says Joseph Chaiken, of course, run a number of capillaries a physical chemist at Syracuse Universiin parallel, but preparing gel-filled cap- ty. Chaiken says he invited Comita to illaries is itself difficult, expensive, and speak at the Physical Chemistry Division time consuming, Smith notes. symposium he organized because the The alternative now being pursued by IBM work impressed him as being excitSmith and Wisconsin coworkers Antho- ing and different. ny J. Kostichka, Michael L. Marchbanks, Scientists have studied extensively the Robert L. Brumley Jr., and Howard laser-induced decomposition of solid Drossman has been to develop an in- thin films for the purpose of patterning strument utilizing ultrathin—on the or- thin metallic films on solid substrates. der of 50 |nm—gel slabs [Bio/Technology, Comita's work, though, appears to be 10, 78 (1992)]. As with capillary gel elec- the first demonstration of the use of a latrophoresis, because the gels are thin, ser to fashion gold conducting lines from high voltages can be applied without gold clusters scattered throughout a damage from heating. polymer matrix. The instrument developed by the Comita and his coworkers, Wolfgang Wisconsin chemists can presently ana- Jacob, Eric Kay, and Rong Zhang, prelyze the products of 18 DNA sequencing pare the metal/polymer composite inreactions in parallel. It utilizes fluores- side a vacuum chamber filled with a cence detection, which is achieved with radio-frequency-generated plasma. Bano moving components using a novel sically, tetrafluoroethylene gas is polydetector design based on a cooled, merized by the plasma on the surface charge-coupled device array detector. of a substrate, producing a polytetraAll 18 samples, each about 600 nano- fluoroethylene (PTFE) film. At the liters in volume and containing about same time, energetic ions striking a 100 ng of DNA, are loaded and detected gold electrode sputter gold atoms into in a region only 1.8 cm wide. the growing PTFE film. The composite Smith stresses that sequencing DNA film typically is grown at the rate of
Gold conducting lines drawn in polymer film
SAN FRANCISCO
APRIL 27,1992 C&EN
39
SCIENCE/TECHNOLOGY
Scanning laser leaves gold line in its wake Laser beam \
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Substrate
about 10Â per second. It takes about 15 minutes or less to grow typical films up to 10,000 À thick. The coated substrate is taken out of the reactor and a scanning visible laser is used to "draw" line patterns on the composite film. The focused laser beam heats the film, causing the polymer matrix to collapse and evaporate. At the same time, the gold clusters in the heated area migrate and coalesce into a continuous film. This gives rise to pure metallic lines as wide as the laser beam— about 5 to 10 μιτι, an appropriate range for microelectronic circuitry. The resis tivity of these gold lines can be made to approach that of bulk gold, Comita says. The gold content of the composite film can be controlled to make a range of films—from totally insulating to con ducting. Comita says this method for generat ing microcircuit patterns is much sim pler than a recently announced method for forming copper lines on PTFE (C&EN, Jan. 6, page 24). The copper-onPTFE process involves three steps: irra diation to form the pattern, chemical etching, and chemical vapor deposition of copper. The IBM process involves only two steps: dry deposition of the composite film and laser writing of the metal line patterns. The second step can be done in ambient air. Furthermore, Comita points out, gold lines conduct as well as copper but are resistant to oxida tion, and thus do not need passivation, as copper does. In addition, the pure polymer films can be subtractively pat terned with a single-step laser exposure. 'The films can thus be built up into a three-dimensional structure with metal lines and/or vias [vertical channels], making this an extremely versatile sys tem for microelectronic circuitry or pack aging applications," he says. 40
APRIL 27,1992 C&EN
The IBM researchers have made some prototype circuit patterns, but no specif ic manufacturing applications are being pursued yet. The cost of using gold, Co mita believes, may not be out of line for "high-end" packaging applications such as mainframes and supercomputers. Syracuse's Chaiken agrees, noting that the small amount of gold needed should help keep the process economical. In any case, Comita believes his patterning pro cess can be extended to other metals, and he is now working to prove that. Ron Dagani
Catalysts span aqueous, organic phase gap
S A N FRANCISCO The search for new catalysts follows many different paths. One of the more intriguing new approaches to catalysis is the development of catalysts that bridge the gap between aqueous and organic phases that are normally active only at their interfaces. A number of catalysts are operative only in aqueous solution, but have po tential applications in petrochemistry. Work aimed at reconciling the usual in tractabilities has begun. Mark E. Davis, a chemical engineering professor at California Institute of Tech nology, has developed a new immobili zation method designed to convert liq uid-phase reactants. At a session of the Division of Industrial & Engineering Chemistry, he described the catalysts as supported-aqueous-phase (SAP) cata lysts, consisting of thin films that reside on high-surface-area hydrophilic sup ports. The film is composed of watersoluble organometallic complexes and water. Reactions of liquid-phase waterinsoluble organics take place at the filmorganic interface. SAP catalysts are distinguished from supported-liquid-phase (SLP) catalysts, in that the SLP catalysts are specifically designed for gas-phase reactants and the SAP catalysts are designed for liq uid-phase reactants. Also, with SLP catalysts, the reaction proceeds homo geneously; with SAP catalysts the reac tion occurs at the interface.
The idea is to impart water solubility to an organometallic complex, known to be a homogeneous catalyst in organic media, by modifying the water solubili ty of its ligands. The water-soluble com plex is supported by a hydrophilic solid and this creates a large interfacial area between catalyst and organic reactants. Thus, being hydrophilic, the ligands and the support provide enough interaction energy to sustain immobilization. An example of the method is the hydroformylation of oleyl alcohol with SAP catalysts at 100 °C and 725 psig of carbon monoxide and hydrogen pres sure, using equal parts of carbon mon oxide and hydrogen. The catalyst was a controlled-pore glass impregnated with HRh(CO)[P(m-C6H4S03Na)3]3 and P(mC6H4S03Na)3 (triphenylphosphine trisulfonate). Water was added to the catalyst from the vapor phase, and the conden sation of the water was terminated after varying lengths of time to permit vary ing the water loading. Extensive experimentation demon strated that the rhodium is not leached into the organic phase. Since neither ole yl alcohol nor the hydroformylation products are soluble in water, the activi ty positively demonstrates that the rho dium is immobilized and that the reac tion occurs at the organic-aqueous inter face. The conversions obtained are proportional to the interfacial area in the reactor. It is necessary for the SAP catalysts to be stable over time. Additional experi ments showed that the components of the SAP catalyst self-assemble in order to function. Under the experimental con ditions, individual components of the SAP catalysts are more stable assembled than separated. The conclusion is that separation of the catalyst during reaction is unlikely. Water content is very influential in catalyst performance. In the hydro formylation of 1-heptene, for example, the turnover frequency rises by two or ders of magnitude as the water content is raised from 2.9% by weight to 9%. The selectivity does not change. Eventually, as the water content is raised still more, the activity reaches a maximum. The rise in the activity is believed to be the result of increased mobility of the complexes. Davis notes that the idea of support ed, aqueous-phase catalysts is rather general and can be applied to a broad range of reactions. In large-scale applica tions there is some concern about the
