Chemistry: molecular biology and microbiology – Plant cell or cell line – per se ; composition thereof;... – Culture – maintenance – or preservation techniques – per se
Reexamination Certificate
2001-10-10
2004-04-06
Ponnaluri, Padmashri (Department: 1639)
Chemistry: molecular biology and microbiology
Plant cell or cell line, per se ; composition thereof;...
Culture, maintenance, or preservation techniques, per se
C435S410000, C435S283100, C435S286100, C435S288200
Reexamination Certificate
active
06716629
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to devices for molecular synthesis, storage and screening, and other chemical, biochemical, biological, and physical experiments, and to methods of making, using, and manipulating the same.
BACKGROUND OF THE INVENTION
High throughput methods for creating and analyzing chemical and biochemical diversity play a vital role in technologies including drug discovery and development. Specific applications of high throughput methods include drug discovery, optimization of reaction conditions (e.g., conditions suitable for protein crystallization), genomics, proteomics, genotyping, polymorphism analysis, examination of RNA expression profiles in cells or tissues, sequencing by hybridization, and recombinant enzyme discovery.
Rapid, high throughput methods for synthesizing (e.g., using combinatorial chemistry methods) and screening large numbers of these compounds for biological and physicochemical properties are desired, for example, to increase the speed of discovery and optimization of drug leads.
Similarly, due in part to the large amount of sequence data from the human genome project, efforts are underway to rapidly obtain x-ray crystallography data for the protein products of many newly discovered genes. One of the rate limiting steps in this process is the search for appropriate solution conditions (e.g., pH, salt concentration) to cause protein crystallization. There is also a need to determine the function of each of the newly discovered genes (i.e., “functional genomics”) and to map protein-protein interactions (i.e., “proteomics”). Given the large number of human genes, protein modifications, and protein binding partners, higher throughput methods are desired.
Another advance in biotechnology is the creation of surfaces with high-density arrays of biopolymers such as oligonucleotides or peptides. High-density oligonucleotide arrays are used, for example, in genotyping, polymorphism analysis, examination of RNA expression profiles in cells or tissues, and hybridization-based sequencing methods as described, for example, in U.S. Pat. Nos. 5,492,806, 5,525,464, and 5,667,972 to Hyseq, Inc. Arrays containing a greater number of probes than currently provided are desirable.
The process of discovering and improving recombinant enzymes for industrial or consumer use has emerged as an important economic activity in recent years. A desire to discover very rare, activity-improving mutations has further stimulated the search for higher throughput screening methods. Such methods often require screening 100,000 to 1,000,000 members of a genetic library in parallel, and then rapidly detecting and isolating promising members for further analysis and optimization.
One of the challenges in the development of high throughput methods is that conventional liquid handling techniques such as pipetting, piezoelectric droplet dispensing, split pin dispensing, and microspritzing are generally unsuitable for rapidly loading or transferring liquids to or from plates of high density (e.g., plates having more than about 384 wells). For example, these techniques can cause substantial splashing, resulting, for example, in contamination of neighboring wells and loss of sample volume. Also, as the number of wells increases, the time necessary to reformat compounds from the previous generation of plates to the higher density plates generally increases, thus limiting the utility of higher density plates. Evaporation can also be problematic with times greater than a few seconds. Moreover, entrapped air bubbles can result in inconsistencies in the loading of small fluid volumes (e.g., less than about one microliter).
Significant bottlenecks in high throughput screening efforts include library storage, handling, and shipping. As the number of compounds in a library increases, the number of 96- or 384-well plates, and the total volume needed to store the libraries, also increases. For compounds that are stored in frozen solvent such as DMSO or water, thawing, dispensing, and refreezing pose the hazard of crystallization, precipitation, or degradation of some compounds, making it difficult to dispense accurate quantities in the future. Having samples stored in low-density plates requires a time consuming step of reformatting the samples into high-density plates before the high-density technology can be utilized.
SUMMARY OF THE INVENTION
The invention features methods of making devices, or “platens”, having a high-density array of through-holes, as well as methods of cleaning and refurbishing the surfaces of the platens. The invention further features methods of making high-density arrays of chemical, biochemical, and biological compounds, having many advantages over conventional, lower-density arrays. The invention includes methods by which many physical, chemical or biological transformations can be implemented in serial or in parallel within each addressable through-hole of the devices. Additionally, the invention includes methods of analyzing the contents of the array, including assaying of physical properties of the samples.
In various embodiments, the reagents can be contained within the through-holes by capillary action, attached to the walls of the through-holes, or attached to or contained within a porous material inside the through-hole. The porous material can be, for example, a gel, a bead, sintered glass, or particulate matter, or can be the inner wall of a through-hole that has been chemically etched. In particular embodiments, the arrays can include individual molecules, complexes of molecules, viruses, cells, groups of cells, pieces of tissue, or small particles or beads. The members of the arrays can also, for example, function as transducers that report the presence of an analyte (e.g., by providing an easily detected signal), or they can function as selective binding agents for the retention of analytes of interest. Using these methods, arrays corresponding to a large plurality of human genes (e.g., using nucleic acid probes) can also be prepared.
On embodiment of the invention features a method of making a platen of a desired thickness having a plurality of through-holes. The method includes the steps of (a) providing a plurality (e.g., 2, 3, 5, 8, 10, 100, 1000 or more) of plates having upper and lower surfaces, wherein one or both of the upper and lower surfaces of at least some of said plurality of plates has continuous, substantially parallel grooves running the length of said surfaces; (b) bonding the upper surfaces of all but one of said plurality of plates to the lower surfaces of the other plates (i.e., the upper surface of the first plate is bonding to the lower surface of the second plate; the upper surface of the second plate is bonded to the lower surface of the third plate; and so on; the upper surface of the last plate is not bonded to anything else); and (c) if necessary to achieve the desired thickness, slicing the platen substantially perpendicularly to the through-holes, thereby creating a platen of a desired thickness having a plurality of through-holes. Step c) can optionally be repeated make a plurality of platens.
The plates can be made from any material that can be bonded (e.g., plastic, metal, glass, or ceramic), and each can have a thickness from, e.g., about 0.01 mm to 2.0 mm, preferably 0.1 mm to 1 mm; the grooves have a depth from, e.g., 0.005 mm to 2.0 mm (i.e., less than the thickness of the plates); and the grooves can have a width from, e.g., 0.1 mm to 1.0 mm.
The plates can be bonded in a configuration in which the grooves of one plate are substantially parallel to the grooves of each of the other plates, or can be bonded so that the grooves of certain plates are perpendicular to, or at acute angles to, the grooves of certain other plates.
In another embodiment, the invention features a device for the immobilization of probes, cells, or solvent. The device includes a platen (optionally having hydrophobic upper and lower surfaces) having a plurality of through-holes (e.g., from the upper surface to the lower surface),
Brenan Colin
Hess Robert A.
Kanigan Tanya S.
Linton John
Ozbal Can
BioTrove, Inc.
Fish & Richardson P.C.
Ponnaluri Padmashri
Tran My-Chau T.
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