Flow cell for synthesis of arrays of DNA probes and the like

Details Flow cell for synthesis of arrays of DNA probes and the like Flow cell for synthesis of arrays of DNA probes and the like

1999-11-10

2001-11-13

Warden, Jill (Department: 1743)

Chemical apparatus and process disinfecting, deodorizing, preser

Control element responsive to a sensed operating condition

C422S091000, C435S006120, C435S007100

Reexamination Certificate

active

06315958

ABSTRACT:

FIELD OF THE INVENTION
This invention pertains generally to the field of biology and particularly to apparatus for use in the analysis and sequencing of DNA and related polymers.
BACKGROUND OF THE INVENTION
The sequencing of deoxyribonucleic acid (DNA) is a fundamental tool of modern biology and is conventionally carried out in various ways, commonly by processes which separate DNA segments by electrophoresis. See, e.g., Current Protocols In Molecular Biology, Vol. 1, Chapter 7, “DNA Sequencing,” 1995. The sequencing of several important genomes has already been completed (e.g., yeast,
E. coli
), and work is proceeding on the sequencing of other genomes of medical and agricultural importance (e.g., human,
C. elegans,
Arabidopsis). In the medical context, it will be necessary to “re-sequence” the genome of large numbers of human individuals to determine which genotypes are associated with which diseases. Such sequencing techniques can be used to determine which genes are active and which inactive either in specific tissues, such as cancers, or more generally in individuals exhibiting genetically influenced diseases. The results of such investigations can allow identification of the proteins that are good targets for new drugs or identification of appropriate genetic alterations that may be effective in genetic therapy. Other applications lie in fields such as soil ecology or pathology where it would be desirable to be able to isolate DNA from any soil or tissue sample and use probes from ribosomal DNA sequences from all known microbes to identify the microbes present in the sample.
The conventional sequencing of DNA using electrophoresis is typically laborious and time consuming. Various alternatives to conventional DNA sequencing have been proposed. One such alternative approach, utilizing an array of oligonucleotide probes synthesized by photolithographic techniques is described in Pease, et al., “Light-Generated Oligonucleotide Arrays for Rapid DNA Sequence Analysis,” Proc. Natl. Acad. Sci. USA, Vol. 91, pp. 5022-5026, May 1994. In this approach, the surface of a solid support modified with photolabile protecting groups is illuminated through a photolithographic mask, yielding reactive hydroxyl groups in the illuminated regions. A 3′ activated deoxynucleoside, protected at the 5′ hydroxyl with a photolabile group, is then provided to the surface such that coupling occurs at sites that had been exposed to light. Following capping, and oxidation, the substrate is rinsed and the surface is illuminated through a second mask to expose additional hydroxyl groups for coupling. A second 5′ protected activated deoxynucleoside base is presented to the surface. The selective photodeprotection and coupling cycles are repeated to build up levels of bases until the desired set of probes is obtained. It may be possible to generate high density miniaturized arrays of oligonucleotide probes using such photolithographic techniques wherein the sequence of the oligonucleotide probe at each site in the array is known. These probes can then be used to search for complementary sequences on a target strand of DNA, with detection of the target that has hybridized to particular probes accomplished by the use of fluorescent markers coupled to the targets and inspection by an appropriate fluorescence scanning microscope. A variation of this process using polymeric semiconductor photoresists, which are selectively patterned by photolithographic techniques, rather than using photolabile 5′ protecting groups, is described in McGall, et al., “Light-Directed Synthesis of High-Density Oligonucleotide Arrays Using Semiconductor Photoresists,” Proc. Natl. Acad. Sci. USA, Vol. 93, pp. 13555-13560, Nov. 1996, and G. H. McGall, et al., “The Efficiency of Light-Directed Synthesis of DNA Arrays on Glass Substrates,” Journal of the American Chemical Society 119, No. 22, 1997, pp. 5081-5090.
A disadvantage of both of these approaches is that four different lithographic masks are needed for each monomeric base, and the total number of different masks required are thus four times the length of the DNA probe sequences to be synthesized. The high cost of producing the many precision photolithographic masks that are required, and the multiple processing steps required for repositioning of the masks for every exposure, contribute to relatively high costs and lengthy processing times.
An improved process for synthesizing arrays of DNA probe sequences, polypeptides, and the like, rapidly and efficiently by a patterning process utilizing a computer controlled image former, is described in published PCT application International Publication No. WO 99/42813, published Aug. 26 1999, entitled Method and Apparatus for Synthesis of Arrays of DNA Probes. This process eliminates the need for a lithographic mask, significantly reducing the costs and time delays that have been associated with processes requiring such masks. In the patterning process described in the foregoing published PCT application, a substrate with an active surface to which, e.g., DNA synthesis linkers have been applied, is used to support probes to be activated. To activate the surface a high precision two-dimensional light image is projected onto the substrate by an image former, illuminating those pixels on the active surface which are to be activated to bind a first base. The light incident on the pixels in the array to which the light is applied deprotects OH groups and makes them available for binding the bases. After this development step, a fluid containing the appropriate base is provided to the active surface of the substrate and the selected base binds to the exposed sites. The process is repeated until all of the elements of the two-dimensional array on the substrate surface have an appropriate base bound thereto. The process is repeated for other pixel locations and desired levels of bases until the entire selected two-dimensional array of probe sequences has been completed. To provide the various chemicals in an appropriate sequence to the substrate, the substrate may be mounted within a flow cell having an enclosure which seals off the active surface of the substrate, allowing the appropriate reagents to flow through the flow cell and over the active surface.
SUMMARY OF THE INVENTION
The present invention is directed to an improved flow cell of the type that may be utilized in the synthesis of arrays of DNA probe sequences, polypeptides and the like, and is particularly adapted to be used with image formers for projecting an array of patterned light onto a substrate held by the flow cell. The flow cell of the invention is formed to precisely align a substrate with respect to an image former while distributing the fluid containing the appropriate chemicals through the active volume and over the active exposed surface of the flow cell, while minimizing the total volume of fluid contained within the flow cell to conserve the reagents being utilized. The flow cell allows fast and simple removal and replacement of substrates while insuring a tight seal around the substrate to minimize the leakage of reagents in the flow cell, and it locates the active surface of the substrate at the focal plane of the image former with a high degree of accuracy and repeatability.
A flow cell of a preferred construction in accordance with the invention includes a base having a central window opening and a registration surface against which a substrate may be mounted with its active surface opposite to that which is engaged against the registration surface. A gasket having a central opening defining an active area surrounded by the material of the gasket is mounted on the active surface of the substrate. The gasket has inlet and outlet extension openings which optionally and preferably extend away from the central opening in the gasket. A press block has an engagement surface which is adapted to the engaged against the gasket to fully enclose an active volume between the press block, the peripheral walls of the central opening in the gasket, and

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