Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
1999-02-17
2001-07-31
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S288400, C435S287200, C435S287300, C435S006120, C422S050000, C422S051000
Reexamination Certificate
active
06268210
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to spatially-addressable sandwiched arrays of compounds, particularly biological compounds such as peptides and polynucleotide probes, and methods of making and using the same. The present invention also relates to a method and device for holding together the individual components composing the sandwich array, more particularly, a clamping device for securely yet safely holding substrates of a sandwich array together during assembly, use, storage, and/or transport of the sandwich array.
BACKGROUND OF THE INVENTION
Recent advances in the ability to construct arrays of biological compounds have greatly facilitated the ease and speed with which certain biological assays can be performed. For example, in the areas of nucleic acid sequencing and analysis, the advent of new technologies for constructing arrays of immobilized target nucleic acids or oligonucleotide probes has enabled the rapid screening and sequencing of nucleic acids. Arrays of peptides and small biomolecules have also proven useful in binding assays used in pharmaceutical development. The usefulness of these arrays depends on the ability to generate and use arrays with spatially addressable regions of defined composition or sequence.
Several technologies have been developed for producing such arrays. For example, several researchers have devised methods for in situ synthesis of arrays of biological polymers, such as nucleic acids, peptides, and carbohydrates. These methods use, for example, physical barriers to separate different synthesis sites, devices (such as inkjet printers) for precise delivery of reagents to different synthesis sites, or masking techniques that allow the use of light to determine the course of synthesis. See, e.g., WO 90/03382; Fodor et al., 1991, Science 251:767-73; Pease et al., 1994, Proc. Natl. Acad. Sci. 91:5022-26; U.S. Pat. No. 5,424,186, to Fodor et al. Alternatively, presynthesized biological compounds or biological polymers may be attached directly to the substrate at precise positions using a variety of techniques, ranging from simple spotting to robotic delivery systems. A variety of different substrates and techniques for attaching the biological compounds to the substrates are also available.
As noted above, arrays of nucleic acids have proven particularly valuable. The ability to perform many previously available techniques has been greatly enhanced by the availability of arrays, which permit many assays to be performed simultaneously, rather than having to do each assay individually. Other techniques that would have been virtually impossible are now possible using polynucleotide arrays.
One technique that has been particularly enhanced by the availability of arrays of nucleic acids is sequencing by hybridization (SBH). SBH is a technique for rapidly sequencing nucleic acids without using gels. In SBH, polynucleotides having overlapping sequences are hybridized to a target nucleic acid. The sequences of the polynucleotides that hybridized are determined and their common sequences overlapped to generate the sequence of the target nucleic acid. The use of arrays has allowed the generation of sufficient hybridization information to make SBH feasible on a large scale.
SBH is divided into three formats, depending on the nature of the array and the way in which it is interrogated. In Format I, an immobilized target nucleic acid is interrogated with labeled solution-phase polynucleotide probes. In Format II, a spatially-addressable array of immobilized polynucleotide probes is interrogated with a labeled solution-phase target nucleic acid. In Format III, an array of immobilized polynucleotide probes is hybridized with an unlabeled solution-phase target nucleic acid and one or more labeled solution-phase oligonucleotide probes. Hybridization is assayed by ligating the labeled oligonucleotide probes to the immobilized polynucleotides. All three formats require the ability to distinguish perfectly matched hybrids from hybrids that contain a single mismatch at any position. For a more detailed discussion of SBH and the three formats, see WO 98/31836, particularly at pages 1-3.
While the availability of high-density arrays of immobilized compounds has revolutionized the speed with which certain biological assays can be performed, array-based assays still suffer from drawbacks. Samples are often available in limited amounts, which are incompatible with the large volumes of assay solutions required to immerse the arrays. Thus, there remains a need in the art for improved arrays that allow the use of small volumes of assay solutions.
SUMMARY OF THE INVENTION
These and other shortcomings in the art are overcome by the present invention, which in one aspect provides spatially addressable sandwich arrays of immobilized compounds. In the sandwich arrays, two or more substrates each having a spatially addressable array of compounds immobilized thereon are combined into “sandwiches” in which the individual arrays are separated by spacer regions. The spacers may be, for example, masks made of TEFLON or other similar, preferably hydrophobic and preferably nonabsorbent, material which may be provided on one or more of the substrates. The masks are designed so that so that when the substrates are pressed together, the masks form at least one reaction chamber with walls defined by the masks and the substrates. Preferably, a plurality of spacers is provided to separate a plurality of arrays on a single substrate; these arrays may be different or may be replicates. The plurality of spacers forms a plurality of chambers in the sandwich array, with each chamber designed to contain a small volume of assay solution. In order to form the desired chamber or chambers, the substrates should be maintained in a fixed position with respect to each other, such as by a holder. Advantageously, the sandwich arrays of the present invention allow two or more compound arrays to be interrogated simultaneously with very small volumes of assay solution.
In a preferred embodiment, the immobilized compounds are immobilized nucleic acid compounds, particularly polynucleotides. However, peptides, proteins, small organic compounds (such as drug candidates), carbohydrates, or any of a variety of other compounds that can be arrayed on a substrate may all be used in the sandwich arrays of the present invention.
In use, the plurality of substrates with a plurality of arrays of compounds attached thereto are held together such that the substrates and spacers form one or more chambers, with the compounds of the arrays exposed inside the chamber or chambers. Assay solution or solutions is added to the chamber or chambers, where it contacts the arrays. The arrays and assay solution(s) are maintained in contact under the desired conditions for the desired time (depending on the requirements of the particular assay being performed). The assay solution(s) is removed. The arrays are optionally washed and separated, or separated and washed. The results of the assay are then determined.
In addition to the sandwich arrays of the present invention, a device is provided for holding together a pair of substrates of a sandwich array and also for serving other desired functions desired during the use of the sandwich array. The device includes a preferably slidable clamping bar having clamping arms biased together to provide sufficient force to securely clamp together the substrates without damaging the substrates. The clamping bar preferably prevents leakage of the solution to be tested, such as by extending across at least the bottom edge. Moreover, the clamping bar preferably has a flat bottom surface to facilitate positioning of the sandwich array in a desired substantially upright orientation for filling. A second clamping bar may be positioned over the end of the substrates opposite the end over which the first clamping bar has been positioned. The clamping walls of the clamping bars of the present invention are preferably dimensioned so that once clamping bars have been positioned over oppo
Baier Joerg
Drmanac Radoje T.
Hauser Brian
Forman B J
Hyseq Inc.
Jones W. Gary
Marshall O'Toole Gerstein Murray & Borun
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