Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
2001-01-31
2003-11-25
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C435S007100, C436S524000, C427S002110
Reexamination Certificate
active
06653151
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to placement of many compounds on a surface in a predefined pattern. Moreover, the present invention discloses uses and methods for manufacture of microarrays having these compounds bound in the predefined pattern to the surface.
BACKGROUND OF THE INVENTION
Synthesis and analysis of large numbers of bound oligonucleotides or peptides are generally known in the art. For example, the Selectide bead approach uses vast quantities of spherical cross-linked polymer beads (Millipore or Cambridge Research Laboratories polyacrylamide beads or Rapp Tentagel polystyrene) divided into 20 equal piles, each of which then has a different L-amino acid coupled to all the beads in the pile. The bead piles are then combined and thoroughly mixed. The resulting single pile is again divided into 20 different piles, each of which is reacted with a different one of the 20 different L-amino acids. This Divide, Couple and Recombine process (DCR) is repeated through six reactions to produce hexapeptides bound to the beads. The beads are then screened against a “target” molecule that is labeled with a conjugated enzyme, such as horseradish peroxidase. The target “sticks” to active hexapeptide(s). The bead is rendered visible by adding a substrate for the enzyme that converts it to a colored dye, which is precipitated within the beads, and then the visually identified bead(s) are picked out with tweezers. The peptides on the beads are then analyzed, for example by the Edman sequencing method, and soluble versions produced in a synthesizer. The initial screening (locating the target bead(s)) takes only days, the makeup of each identified hexapeptide is unknown, and the analysis and synthesis for confirmation and further work takes much longer. Such sorting and resorting becomes too burdensome and labor intensive for the preparation of large arrays of peptides. Further, this process can be characterized as not calling for a continuous support, and it is not addressable.
Another approach, using arrays, is the pin dipping method for parallel oligonucleotide synthesis. Geysen, J. Org. Chem. 56, 6659 (1991). In this method, small amounts of solid support are fused to arrays of solenoid controlled polypropylene pins, which are subsequently dipped into trays of the appropriate reagents. The density of arrays, however, is limited, and the dipping procedure employed is cumbersome in practice.
Disclosed at the Southern, Genome Mapping Sequence Conference, May 1991, Cold Spring Harbor, N.Y., is a scheme for oligonucleotide array synthesis in which selected areas on a glass plate are physically masked and the desired chemical reaction is carried out on the unmasked portion of the plate. The problem with this method is that it is necessary to remove the old mask and apply a new one after each interaction. Fodor et al., Science 251, 767 (1991) describes another method for synthesizing very dense 50 micron arrays of peptides (and potentially oligonucleotides) using mask-directed photochemical de-protection and synthetic intermediates. This method is limited by the slow rate of photochemical de-protection and by the susceptibility to side reactions (e.g., thymidine dimer formation) in oligonucleotide synthesis. Khrapko et al., FEBS Letters 256, 118 (1989) suggest simplified synthesis and immobilization of multiple oligonucleotides by direct synthesis on a two-dimensional support, using a printer-like device capable of sampling each of the four nucleotides into given dots on the matrix. For example, the probes are applied to a chip with a pin or a pipette in the pattern of an array and immobilized by any of a variety of techniques such as adsorption or covalent linkage. An example of such DNA arrays is described in Stimpson et al. Proc. Natl. Acad. Sci. USA Vol. 92, pp. 6379-6383, July 1996. Since elements of the array are formed by the application of a DNA solution to the surface of the array the process is relatively slow. The development of VLSIPS.TM. technology has provided methods for making very large arrays of oligonucleotide probes in very small arrays. See U.S. Pat. No. 5,143,854 and PCT patent publication Nos. WO 90/15070 and 92/10092. U.S. patent application Ser. No. 082,937, filed Jun. 25, 1993, describes methods for making arrays of oligonucleotide probes that can be used to provide the complete sequence of a target nucleic acid and to detect the presence of a nucleic acid containing a specific nucleotide sequence. One drawback to this method is that it relies on a new DNA synthesis chemistry as opposed to the standard phosphoramidite chemistry used in commercial DNA synthesizers. The technology feeds off the methods evolved in the electronics industry and therefore has some of the same requirements, vis, accurate positioning to micron scales, clean room requirements and the use of multiple photo-masks to define the array pattern. Although electronic “chips” (for example an Intel Pentium.RTM. microprocessor) are mass-produced economically, they are typically too expensive to be used as a disposable element, as is needed with a DNA chip.
A common limitation to many of these methods is due to depositing liquids on surfaces, i.e., “spreading.” For example, spreading occurs on derivatized surfaces, such as those used in DNA immobilization on glass supports, because the solid support surface becomes hydrophilic upon derivatization. As a result, when the DNA (desired to be immobilized upon the solid support) is contacted with the surface of the solid support, it spreads, rather than remaining in a discrete “spot.” Spreading is a major constraint on array density (i.e., the number of different spots that can be arranged on a single solid support). Hence, any means to curtail spreading, and so increase array density, is highly desirable.
Additional problems arise with the density of biomolecule spotted on the solid support. Droplets of liquid will form a meniscus, which inherently causes uneven liquid thickness and the edges will dry at a different period of time from the center of the droplet. Thus, the coverage of biomolecule on the surface remaining may be uneven.
Still further when forming a microarray by spotting technology, the total amount of biomolecule deposited on the region of the microarray is limited to the maximum amount soluble in the droplet. For insoluble or low solubility molecules, this becomes a limiting factor.
Unfortunately, all of the array fabrication methods mentioned above also suffer from the same general problem in that each element of each array is a unique synthesis or an application step. This is true even when array elements or entire arrays are simply duplicated or produced “in parallel”, or more accurately, concurrently. Since each element is a unique synthesis or application there is a chance for variation between corresponding elements on different arrays or, for that matter, duplicated elements on the same array. Even in a photolithographic process, increasing the number of chips on a wafer (the substrate on which multiple arrays are produced) results in an increase in surface area, which increases demand on the chemicals used in photochemistry (assuming no change in chip size).
What is needed in the art are methods to enhance the amount of material that attaches to a solid support and to increase the reliability and reproducibility with which materials are applied to a solid support. The present invention helps meet that need.
Biochemical molecules on microarrays have been synthesized directly at or on a particular cell on the microarray, or preformed molecules have been attached to particular cells of the microarray by chemical coupling, adsorption or other means. The number of different cells and therefore the number of different biochemical molecules being tested simultaneously on one or more microarrays can range into the thousands. Commercial microarray plate readers typically measure fluorescence in each cell and can provide data on thousands of reactions simultaneously thereby saving time and labor. A representative exam
Anderson N. Leigh
Anderson Norman G.
Braatz James A.
Counts Gary
Large Scale Proteomics Corporation
Le Long V.
Roylance Abrams Berdo & Goodman L.L.P.
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