Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
1999-10-29
2001-12-11
Ponnaluri, Padmashri (Department: 1627)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C530S333000, C530S334000, C536S063000, C536S025300, C422S129000, C422S131000, C422S134000, C435S283100, C435S286500, C435S288400, C435S292100, C435S091500, C435S091500, C435S091500, C435S091500, C435S091500
Reexamination Certificate
active
06329210
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of Use
The invention is directed towards the automated synthesis of polymers. More specifically, the invention relates to an apparatus for serial, continuous polymer synthesis, capable of rapid and low cost production of a large number of oligonucleotides.
2. Field of Use
Articles and publications set forth in this patent disclosure are presented for the information contained therein; none of the information is admitted to be statutory prior art and we reserve the right to establish prior inventorship with respect to any such information.
Oligonucleotides are used extensively in modem biotechnology. Applications for oligos include investigations relating to gene expression, polymorphism, drug discovery, and diagnostics, to name a few.
Arrays require large number of oligos, on the average of 10,000 per array. Oligo synthesis is vitally important for investigations using arrays. Oligos may be synthesized in situ, or “grown” where they are subsequently to be use, or oligos synthesized offline, deposited and attached to an array substrate. Such offline synthesis is commonly referred to as the synthesis of “whole” oligos. While automated DNA synthesizers exist, the time required to produce 10,000 oligos for an array is hundreds of hours. A batch type process and apparatus (see, for example, U.S. Pat. No. 5,814,700) can produce 8 dozen 25 mer oligo about every 2.5 hours. Shorter or longer lengths of oligos may be produced, but the batch process does not facilitate dramatic compression of synthesis times. Moreover, automatic synthesizers in use are not amenable to producing small amounts of oligos. Automatic synthesizers typically produce on the order of 200 Pico moles of an oligo, which is enough oligo for more than 400,000 arrays! Few applications require that many identical arrays. Often the actual amount required may be 40 nanomoles or less. The minimum quantities produced by automatic synthesizers result in much wasted oligos and all materials, included reagents and solvents, used in oligo synthesis.
Synthesizers such as the ABI, use columns as the support for the synthesis. Typical machines can support one to four columns at a time. Some others can have up to twenty-four in parallel. These systems are a closed flow-through operation All reagents are tied together to a common manifold. Both time and reagent are spent to flush our prior reagents switch to another reagent, and flow enough material through the column. Batch operation machines, such as described by Brennan (U.S. Pat. No. 5,814,700) use an array of wells, such as a 96 well titer plate. A solid support is inserted into each well and reagents are dropped into each well, and a differential pressure is applied from the top to the exit port at the bottom of the wells. The solution flows through the solid support to waste. Each line is dedicated to a single reagent, thus ensuring that the required amount will be delivered to the wells. A disadvantage is the time required dispensing reagent to each of the wells. Although reagent can be dispensed in parallel by replicating dispensers, the system then bears added complexities. Moreover, in apparatus in which reagents are dispensed serially, dwell times are different for the wells at the beginning of the titer plate versus wells at the end. Exposure times are not identical across rows. All synthesis chemistry is not equally tolerant of such differences, and the synthesis may be adversely affected.
Further, in batch mode, addition of each reagent must be complete before removal may be commenced.
As concerns scaling up the oligo synthesis process, merely adding more wells to a batch process, while increasing throughput, does not increase productivity. The more wells, the more time required to add reagents, or, alternatively, the more added reagent dispensing capability increases the complexity of the system.
At the most practical level, adding more reagent addition nozzles is hindered by close array spacing in denser arrays. Although approaches, such as adding a Y actuator, could be added to denser arrays, dispensing and actuation time become increasingly problematic.
What is needed is a scalable oligo synthesis apparatus capable of quick and cost effective oligo synthesis suitable for use in applications such as arrays.
SUMMARY OF THE INVENTION
The invention provides a scalable synthesis apparatus capable of quick and cost effective oligo synthesis , such oligos suitable for use in applications such as arrays.
The invention provides a polymer synthesis apparatus for building a polymer chain by sequentially adding polymer units. The polymer synthesis apparatus is a serial-based operation machine. The apparatus provides several stations. Each station contains a series of stopping locations, each stopping capable of performing a step in the synthesis protocol. A continuous strip moves through the apparatus, operable to present the stopping locations with solid support sites. Each site of the solid support is either on, encapsulated in, or is otherwise a location on a continuous strip of material, a strip containing sites for synthesis operations, that is moving continuously through the synthesizer. Each functional synthesis site on the strip has a vacuum egression operable to provide an exit for reagents and gases introduced from the apparatus and through an ingress into the sealed site. As the solid support strip moves to the next stop position, the locations for the synthesis on the strip are directly under the locations for a particular operation. Stations as well as stopping locations may be added or removed to adapt the apparatus for the particular synthesis application, which may be a longer or shorter oligo. Briefly, a particular synthesis may require twelve stopping locations. As a strip is passing through the station, the first location for synthesis stops under a location for deblocking. The station actuates the deblocking by making a seal on the strip around the site for synthesis. The deblocking solution is allowed to flow across the synthesis membrane removing the DMT or other protecting group on the membrane. The flow of the deblocking solution is stopped and seal is removed from the strip. Then, the entire strip moves to the next stopping position. The site that was just under the deblocking station is now under the Wash-after-deblocking station and a new site on the strip is now under the deblocking station. A seal is now made for both locations and deblocking occurs in the second synthesis location. In the first synthesis location, an inert gas is passed through the membrane to remove the remaining deblocking reagent from the first location and then is cleaned by passing Acetonitrile (ACN) through the membrane and then inert gas again to dry the membrane. The cleaning step in the second location and the deblocking step in the first location are performed in parallel. When both are finished, the seal is broken again and the strip advances to the next indexed position. The parallel step and indexed progression proceed until the strip has passed through all the stations and synthesis protocols have been completed at each synthesis location on the strip. The steps involved include indexing the synthesis strip to the next location, creating a seal to the synthesis site, and causing the appropriate reagent and/or gases to pass through the membrane. As the first synthesis site on the strip passes the last location on the station, all of the required operations have been performed to add one monomer to the synthesis site. A plurality of synthesis sites, L
1
to LN, may be provided in an advanceable manner by moving the continuous strip. Both the synthesis sites L
1
to LN and the reagent introduction stations S
1
to SN are aligned by movement of the continuous strip. After the step of unsealing, the synthesis sites are advanced by moving the continuous strip until the next reagent introduction station is in alignment whereupon the sealing member of that reagent introduction station is then sealably coupled or engaged to the synt
Agilent Technologie,s Inc.
Ponnaluri Padmashri
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