Chemistry: analytical and immunological testing – Automated chemical analysis – With aspirator of claimed structure
Reexamination Certificate
2001-12-07
2004-02-24
Alexander, Lyle A. (Department: 1743)
Chemistry: analytical and immunological testing
Automated chemical analysis
With aspirator of claimed structure
C436S043000, C436S048000, C436S180000, C422S105000, C422S151000, C073S863310, C222S145300
Reexamination Certificate
active
06696298
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates generally to solid phase processing and, more particularly, to an apparatus for dispensing reagents and other fluids to a plurality of reaction sites for solid phase processing including solid phase synthesis of complex chemicals such as oligonucleotides and the like.
A variety of separative, synthetic, and enzymatic or otherwise catalytic processes use beds of particulate material with transport of reactants, reagents and products or eluants in solution through the bed. In addition, many reactions are known in which the products are separated by concentration in one of two or more phases. These processes include, among others, ion exchange chromatography, gel filtration, ion exclusion chromatography, affinity chromatography, separations based on hydrophobicity, purification based on hybridization, peptide synthesis, oligonucleotide synthesis, and polysaccharide synthesis including combinations of the last three. These processes may be carried out on a small scale for analytical purposes or process design, and are then often scaled up for preparative work. In nearly all examples the solid phase particulates are packed in a closed column with a porous frit on the lower end, an optional frit at the top, and with fluid-connections at both ends so that liquid can flow in either direction through the bed. To achieve efficiency and high resolution with solid phase supports, all volume elements of all fluids should flow through paths of identical composition and nearly identical length, and all particles in the bed should be exposed to the same succession of liquids under the same conditions.
In solid phase systems, some interaction occurs between the solutes running through the bed and the particles composing the bed. This interaction may be based on secondary forces (ionic, hydrophobic, or on immunochemical interactions, or base pairing) or primary valencies as when amino acids or nucleotides are added to a growing chain on the solid phase support, or when immobilized enzymes cleave substrates flowing through the bed, or when enzymes in solution react with substrates attached to the packing. In addition, solvents or reagents of successively differing composition which dissociate adsorbed or otherwise attached bound molecular species, or which cleave off protective groups, or compounds including polymers which have been synthesized on the support may be made to flow through the support. The dissociated or cleaved substances then are free to flow out of the bed in flowing liquid.
In particular, nucleic acid synthesis (generally referred to as “DNA synthesis”) is the process of constructing synthetic single-stranded oligonucleotide through linking of nucleotide, the basic building blocks for DNA. In an automated system, the various steps are carried out by a reagent delivery system which dispenses a number of chemical reagents in a predetermined sequence in a cycle into a synthesis reaction column containing the solid-phase support, according to instructions from the system controller or computer. After the desired number of cycles have been completed, the synthesized oligonucleotide is separated from the reaction column and collected in a vial. This step is generally referred to as “cleavage”. The oligonucleotide may further be subject to a step generally referred to as “deprotection” to complete isolation of the oligonucleotide. In a process for synthesizing polynucleotides on a solid support, the solid support traditionally consists of glass beads of controlled porosity (CPG) or, more generally, of particles of a functionalized inorganic or organic polymer.
The isolation of oligonucleotide involves the treatment of the solid bound oligonucleotide with a cleavage and/or deprotection reagent. Typically, this reagent is concentrated ammonia solution in water but can be other homogeneous or heterogeneous solutions of appropriate bases, alcohols and water. The cleavage and deprotection process is typically performed in two steps. The cleavage of the oligonucleotide is performed at room temperature for approximately one hour before decanting the mixture into a pressure-sealable vessel for extended higher temperature treatment to effect the removal of secondary protecting groups on the synthetic oligonucleotide. This two step process reduces the quantity of support related contaminants in the final isolated product.
The use of a single nozzle for delivering different reagents into a reaction site, well, or column is not feasible because the nozzle will need to be cleaned or flushed out between reagents to avoid contamination, resulting in a high cost and a low throughput. In one conventional chemical synthesis system, a plurality of reagent dispensing nozzles are arranged in a linear array, and the plate containing the reaction cell(s) or column(s) is moved under the linear array to receive reagents from the dispensing nozzles one at a time. The throughput remains low.
Another synthesis apparatus is disclosed in U.S. Pat. Nos. 5,814,700, 5,837,858, and 6,001,311 employing an array of nozzles. A transport mechanism aligns the reaction wells and selected nozzles for deposition of the liquid reagent into the selected reaction wells. Elaborate manipulation of the transport mechanism is used to dispense reagents from the various nozzles into the various reaction wells in sequence to provide simultaneous synthesis in the reaction wells. The throughput is still relatively low because each nozzle can dispense only one reagent.
BRIEF SUMMARY OF THE INVENTION
Embodiments of the present invention are directed to an improved chemical synthesis apparatus for performing chemical synthesis such as nuclei acid synthesis in a plurality of reaction wells or cells in an efficient manner. The apparatus employs dispenser heads that each include a cluster of nozzles which are fluidicly coupled to a plurality of reagent sources for dispensing different reagents through the single dispenser head. Because each dispenser head is capable of dispensing a plurality of different reagents, the apparatus can perform simultaneous synthesis in a plurality of cells at a high throughput without complex and elaborate control of movement of the dispenser heads relative to the cells.
In accordance with an aspect of the present invention, a multi-channel reagent dispenser head for introducing a plurality of reagents into a reaction site comprises a dispenser head body having a dispensing end which is configured to dispense a plurality of reagents from a plurality of reagent sources. A group of nozzles include a plurality of reagent dispensing nozzles which are fluidicly coupled with the plurality of reagent sources. The group of nozzles are clustered to provide a plurality of nozzle outlets in the dispenser head body to introduce reagents from the plurality of reagent sources through the dispensing end of the dispenser head body into the reaction site.
In some embodiments, the plurality of reagent dispensing nozzles are each separately coupled with one of the plurality of reagent sources. The plurality of reagent dispensing nozzles may be separately coupled with reagent sources of building block elements such as bases A, C, G, T, and an activator such as tetrazole. Alternatively, the plurality of reagent dispensing nozzles may be separately coupled with reagent sources of acid deblock, oxidizers, and capping agents. The group of nozzles desirably include a wash nozzle which is fluidicly coupled with a source of wash solvent. The wash solvent may comprise acetonitrile. The group of nozzles desirably include a vacuum nozzle which is coupled to a vacuum source. The nozzle outlet of the vacuum nozzle may be disposed proximal of the nozzle outlets of the reagent dispensing nozzles. In specific embodiments, the dispensing end of the dispenser head body has a maximum dimension of about 9 mm. The nozzles each have an outer diameter of less than about {fraction (1/16)} inch.
In accordance with another aspect of the present invention, a multi-channel reagent dispenser apparatus f
Baltzley Arnie
Cook Ronald M.
Dill Rand
Alexander Lyle A.
Biosearch Technologies, Inc.
Townsend and Townsend / and Crew LLP
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