Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-04-24
2003-12-23
Fredman, Jeffrey (Department: 1637)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S022100, C536S023100, C530S350000, C530S387100
Reexamination Certificate
active
06667394
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to the synthesis and placement of materials at known locations. In particular, one embodiment of the invention provides a method and associated apparatus for the selective application of an array of oligonucleotides on a substrate by way of standard dimethoxytrityl (DMT) based chemistry. The invention may be applied in the field of preparation of an oligomer, a peptide, a nucleic acid, an oligosaccharide, a phospholipid, a polymer, or a drug congener preparation, especially to create sources of chemical diversity for use in screening for biological activity.
Industry utilizes or has proposed various techniques to synthesize arrays of oligonucleotides. One such technique is the use of small rubber tubes as reaction chambers to make up a single dimensional array by the sequential addition of reagents. This technique has advantages by the use of standard DMT based chemistry. However, a limitation with resolution often exists with such technique. Typically the smallest cell size is about 1 millimeter in dimension. This method also does not enable the synthesis of a sufficiently large number of polymer sequences for effective economical screening. A further limitation is an inability to form an array of, for example, oligonucleotides at selected regions of a substrate.
Other representative techniques are described in U.S. Pat. No. 5,143,854 and WO93/09668 which is hereby incorporated by reference for all purposes. Such techniques are finding wide use and are considered pioneering in the industry. In some applications, however, it is desirable to have alternative techniques and chemistries for synthesis of compound libraries.
It would be desirable to have a method and apparatus for making high density arrays of oligonucleotides using DMT-based chemistry and other suitable oligonucleotide synthesis chemistries, as is a method and apparatus for conventional phosphoramidite-based synthesis of a spatially defined array of oligomers (e.g., polynucleotides, polypeptides, oligosaccharides, and the like) each having a substantially predetermined sequence of residues (i.e., polymerized monomer units).
SUMMARY OF THE INVENTION
According to the present invention, a method and apparatus to form an array of polymers, such as oligonucleotides and a substrate using conventional linkage chemistries (e.g., standard DMT-based oligonucleotide synthesis chemistry) is provided. The method and apparatus includes use of selected printing techniques in distributing materials such as barrier materials, deprotection agents, base groups, nucleosides, nucleotides, nucleotide analogs, amino acids, imino acids, carrier materials, and the like to selected regions of a substrate. Each of the printing techniques may be used in some embodiments with, for example, standard DMT-based chemistry for synthesis of oligonucleotides, and in particular selected deprotecting agents in vapor form.
In a specific embodiment, the present invention provides a method of forming polymers having diverse monomer sequences on a substrate. In an embodiment, the method is used to synthesize oligonucleotides having predetermined polynucleotide sequence(s) on a solid substrate, typically in the form of a spatially defined array, wherein the sequence(s) of an oligonucleotide is positionally determined. The present method includes steps of providing a substrate with a linker molecule layer thereon. The linker molecule layer has a linker molecule and a protective group. The present method also includes a step of applying a barrier layer overlying at least a portion of the linker molecule layer. The barrier layer shields the underlying portion from contact with a reagent capable of otherwise reacting with the underlying portion and applied subsequent to application of the barrier layer, thereby substantially precluding a predetermined chemical reaction from occurring on areas of the substrate overlaid with the barrier material. The applying step forms selected exposed regions of the linker molecule layer. A step of exposing the selected exposed regions of the linker molecule layer (e.g., regions not overlaid with the barrier material) to a reagent, typically in vapor phase, and often comprising a deprotecting agent is also included.
In an alternative specific embodiment, the present method includes a method of applying a medium in selected regions of a substrate. The present method includes steps of providing a substrate with a top surface, and selectively applying a medium having an element selected from a group consisting of a barrier material, a receptor, a deprotection agent, a monomer group, a carrier material, and an activator to selected regions of the substrate top surface.
In an embodiment, the invention provides a method for synthesizing a spatial array of polymers of diverse monomeric sequence (e.g., such as a collection of oligonucleotides having unique sequences), wherein the composition (e.g., nucleotide sequence) of each polymer is positionally defined by its location in the spatial array. In general, the method employs a masking step whereby a spatially distributed barrier material is applied to a substrate to block at least one step of a monomer addition cycle from occurring on a portion of the substrate overlaid by the barrier material. The method comprises applying a barrier material to a first spatially defined portion of a substrate, said substrate optionally also comprising a layer of linker molecules and/or nascent polymers (e.g., nascent oligonucleotides), whereby the barrier material overlaying said first spatially defined portion of said substrate shields the underlying portion from contact with a subsequently applied reagent capable of otherwise reacting with the underlying portion and necessary for a complete monomer addition cycle whereby a monomer unit is covalently linked to a nascent polymer or linker, thereby substantially precluding a chemical reaction from occurring on said first spatially defined portion which is overlaid with the barrier material and providing a remaining unshielded portion of said substrate (i.e., portion(s) not overlaid with the barrier material) available for contacting said subsequently applied reagent and undergoing said chemical reaction necessary for a complete monomer addition cycle (i.e., polymer elongation). The subsequently applied reagent is typically a monomer (e.g., nucleotide, nucleoside, nucleoside derivative, amino acid, and the like), a deprotecting agent for removing protecting group(s) which block polymer elongation (e.g., removal of DMT groups by acid hydrolysis), a coupling agent (e.g., phosphoramidites, such as cyanoethyl phosphoramidite nucleosides), a capping agent (e.g., acetic anhydride and 1-methylimidazole), and/or an oxidation agent (e.g., iodine; such as in iodine:water:pyridine:tetrahydrofuran mixture). The method further provides that, subsequent to the application of the barrier material, the reagent(s) is/are applied and permitted to chemically react with the unshielded portion of the substrate for a suitable time period and under suitable reaction conditions. Following reaction of the unshielded portion with the reagent(s), monomer addition is completed and the barrier material is removed (not necessarily in that order), resulting in a monomer addition to polymer(s) in the unshielded portion of the substrate and substantial lack of monomer addition to polymer(s) in the shielded portion of the substrate, during said monomer addition cycle.
In an embodiment, the masking step, wherein a barrier material is applied to a spatially defined portion of the substrate and used to shield said spatially defined portion to block a monomer addition cycle on said spatially defined portion, is employed repetitively. A first barrier mask is applied to overlay a first spatially defined portion of a substrate creating: (1) a first shielded portion overlain by said barrier mask, and (2) a first unshielded portion comprising the portion of the substrate not overlain by said barrier mask. The application of the first b
Fodor Stephen P. A.
Goldberg Martin J.
Goss Virginia
McGall Glenn
Pease R. Fabian
Affymetrix Inc.
Banner & Witcoff , Ltd.
Fredman Jeffrey
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