Photolithography aids DNA research
By Susan English-Seaton
Biotechnology
Santa Clara, CA — Affymetrix is now marketing its GeneChip technology — a unique blend of photolithography and combinatorial chemistry — to produce a nucleic acid analysis system that recognizes sequences of DNA, troubleshooting for the mutated genes that cause cancer and AIDS. The basic concept is to convert living chemistry into data capturable by computer, using the inherent propensity of one DNA strand to match with another of the complementary sequence to recognize and track mutations. Affymetrix`s GeneChip HIV PRT array performs sequence analysis to detect patterns of mutations that can impact the effect of anti-viral drugs such as those used to combat AIDS.
Instead of silicon wafers, 5-inch glass chips — DNA probes arrays containing as many as 65,000 smaller glass chips — are used to chemically recognize the real gene so precisely that it reveals how actively the gene has been working and what mutations it carries. This helps the system track mutations in the AIDS virus as it develops resistance to drugs. Such tracking implies the ability to screen whole populations at risk.
Although not the only company in Silicon Valley to use resequencing as a tool to match targeted genes, Affymetrix is the only one that uses semiconductor fabrication techniques to create the glass chips which do the actual resequencing. Just as particulate contamination is harmful to semiconductor processing, it can also destroy or mar the features of the GeneChip, inhibiting its ability to recognize the correct match.
The system consists of the GeneChip Probe Array, containing hundreds of thousands of different oligonucleotide probes; GeneChip Reagents; the GeneChip Fluidics Station 40, which automates the hybridization of the target to the array; the GeneChip Scanner 50, which uses an argon-ion laser to excite fluorescent molecules incorporated into the target bound to the probe array; and, according to Bob Carroll, Affymetrix`s senior director of manufacturing operations, one of the key ingredients of the system — the GeneChip software. During scanning, an image of the probe array is displayed in real time, and fluorescence intensity information is automatically stored in a raw data file. Following image acquisition, software algorithms use average intensity values from different probe cells to generate genetic information, which can be used for sequence analysis, genotyping and gene expression monitoring.
The process
Basically, a photo-protected glass substrate is selectively illuminated by light passing through a photolithographic mask. Deprotected areas are activated, and with nucleoside incubation, chemical coupling occurs at activated positions. A new mask pattern is then applied and the coupling step repeated. The whole process is repeated until the specified set of probes is produced. The wafers are diced, and individual probe arrays are packaged in injection-molded plastic cartridges, which protect them from the environment and also serve as chambers for hybridization. After the hybridization reaction is complete, the array is inserted into the scanner, where patterns of hybridization are detected. Probes that perfectly match the target generally produce stronger signals than those that have mismatches. Since the sequence and position of each probe on the array are known, the identity of the target nucleic acid applied to the probe array can be determined.
In a large room rated at better than Class 100,000, under a Class 100 laminar flow hood, the preparation process takes place. Basic particle counts and daily wipedowns are procedure, and employees wear hair or beard nets, smocks and foot coverings. Glass substrates are dipped in a series of acid baths and DI water, then coated for surface prep. Says Carroll: “What we`re doing is basically taking a piece of glass and laying down one of the four bases of DNA, then using photolithography to knock off a protecting group at certain pre-defined locations. Then you put another layer of chemistry down.”
Probes are generally built up to about 20 bases. The wafer is then cut up into squares of 49, 169 or 400 pieces (multipiles of grids measuring 7 ¥ 7, 13 ¥ 13, 10 ¥ 10 or 20 ¥ 20). Says Carroll, “The biggest danger from contamination is when you`re actually building the probes on the surface of the glass. If the environment is too dirty, some features could end up missing.” Equipment that actually is used to build the probes is located inside the minienvironment, which features a HEPA-filtered ceiling. Individual chips, or probe arrays, are then put into a plastic package and glued in. The customer takes the probe array (the entire cartridge) and injects a fluorescently tagged sample into it — one that`s been tagged for the application the chip has been designed for. Laser scanning activates the fluorescent tags, which light up. Then the software identifies how bright it is and the location. The grid supplies the map which the software analyzes.
Market driver
Like the semiconductor industry, smaller feature sizes are the goal. “What limits the ability to go down to a smaller and smaller feature size is the scanning technology,” Carroll says. Affymetrix has a collaboration with Hewlett-Packard, which has just produced a state-of-the-art 20-micron reading scanner. “We have basically two types of customers: one group wants more and more information on a given chip; the other wants lower and lower cost chips. Both of these are driven by feature size.” Originally, feature size was 400 microns. Currently, Affymetrix is producing 50-micron features, cramming 65,000 probes on a chip. By the end of the year, the company hopes to move to a 20-micron size, which will accommodate 400,000 probes. Says Bob Carroll: “We`re driving the feature size down, and obviously it will get smaller and smaller, and maybe the wafers will get bigger and bigger.”