This invention relates to chambers for carrying out procedures such as the preparation of substrates having a plurality of reaction spots. The invention has particular application in the manufacture of supports having bound to the surfaces thereof a plurality of chemical compounds, such as biopolymers, which are prepared on the surface in a series of steps.
In the field of diagnostics and therapeutics, it is often useful to attach species to a surface. One important application is in solid phase chemical synthesis wherein initial derivatization of a substrate surface enables synthesis of polymers such as oligonucleotides and peptides on the substrate itself. Support bound oligomer arrays, particularly oligonucleotide arrays, may be used in screening studies for determination of binding affinity. Modification of surfaces for use in chemical synthesis has been described. See, for example, U.S. Pat. No. 5,624,711 (Sundberg), U.S. Pat. No. 5,266,222 (Willis) and U.S. Pat. No. 5,137,765 (Farnsworth).
Determining the nucleotide sequences and expression levels of nucleic acids (DNA and RNA) is critical to understanding the function and control of genes and their relationship, for example, to disease discovery and disease management. Analysis of genetic information plays a crucial role in biological experimentation. This has become especially true with regard to studies directed at understanding the fundamental genetic and environmental factors associated with disease and the effects of potential therapeutic agents on the cell. Such a determination permits the early detection of infectious organisms such as bacteria, viruses, etc.; genetic diseases such as sickle cell anemia; and various cancers. This paradigm shift has lead to an increasing need within the life science industries for more sensitive, more accurate and higher-throughput technologies for performing analysis on genetic material obtained from a variety of biological sources.
Unique or misexpressed nucleotide sequences in a polynucleotide can be detected by hybridization with a nucleotide multimer, or oligonucleotide, probe. Hybridization is based on complementary base pairing. When complementary single stranded nucleic acids are incubated together, the complementary base sequences pair to form double stranded hybrid molecules. These techniques rely upon the inherent ability of nucleic acids to form duplexes via hydrogen bonding according to Watson-Crick base-pairing rules. The ability of single stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) to form a hydrogen bonded structure with a complementary nucleic acid sequence has been employed as an analytical tool in molecular biology research. An oligonucleotide probe employed in the detection is selected with a nucleotide sequence complementary, usually exactly complementary, to the nucleotide sequence in the target nucleic acid. Following hybridization of the probe with the target nucleic acid, any oligonucleotide probe/nucleic acid hybrids that have formed are typically separated from unhybridized probe. The amount of oligonucleotide probe in either of the two separated media is then tested to provide a qualitative or quantitative measurement of the amount of target nucleic acid originally present.
Direct detection of labeled target nucleic acid hybridized to surface-bound polynucleotide probes is particularly advantageous if the surface contains a mosaic of different probes that are individually localized to discrete, known areas of the surface. Such ordered arrays containing a large number of oligonucleotide probes have been developed as tools for high throughput analyses of genotype and gene expression. Oligonucleotides synthesized on a solid support recognize uniquely complementary nucleic acids by hybridization, and arrays can be designed to define specific target sequences, analyze gene expression patterns or identify specific allelic variations. The arrays may be used for conducting cell study, for diagnosing disease, identifying gene expression, monitoring drug response, determination of viral load, identifying genetic polymorphisms, analyze gene expression patterns or identify specific allelic variations, and the like.
In one approach, cell matter is lysed, to release its DNA as fragments, which are then separated out by electrophoresis or other means, and then tagged with a fluorescent or other label. The resulting DNA mix is exposed to an array of oligonucleotide probes, whereupon selective binding to matching probe sites takes place. The array is then washed and interrogated to determine the extent of hybridization reactions. In one approach the array is imaged so as to reveal for analysis and interpretation the sites where binding has occurred. Arrays of different chemical compounds or moieties or probe species provide methods of highly parallel detection, and hence improved speed and efficiency, in assays. Assuming that the different sequence polynucleotides were correctly deposited in accordance with the predetermined configuration, then the observed binding pattern will be indicative of the presence and/or concentration of one or more polynucleotide components of the sample.
The arrays may be microarrays created by in-situ synthesis of biopolymers such as polynucleotides, polypeptides, polysaccharides, etc., and combinations thereof, or by deposition of molecules such as oligonucleotides, cDNA and so forth. In general, arrays are synthesized on a surface of a substrate by one of any number of synthetic techniques that are known in the art. In one approach to the synthesis of microarrays, an apparatus is employed that comprises a reaction chamber and a device for dispensing reagents to the surface of a substrate at discrete sites. A positioning system, which may be a robotic manipulator, moves the substrate to the chamber, in which the device for dispensing reagents is housed. Alternatively, the device for dispensing reagents may be moved in and out of the chamber. A controller controls the application of the reagents to the substrate according to predetermined procedures.
In situ syntheses generally require a controlled environment in the reaction chamber. For example, many syntheses require an anhydrous environment to avoid the destructive effects of exposing chemical reagents to humidity present in the ambient atmosphere. Typically, an anhydrous chamber is created by enclosing the device for dispensing reagents in a reaction chamber through which dry gas is purged. At least a portion of the dispensing apparatus as well as the substrate, to the surface of which dispensed reagents are to be applied, are enclosed in a chamber. Also included are the various stages such as x,y stages used to move the substrate relative to the dispensing apparatus and allowing the desired reagents to be applied to predetermined locations.
To produce arrays it is important to reproducibly perform reactions at a particular site without affecting adjacent sites. The reaction should approximate stoichiometry in producing the desired product. Since many of the reactions are performed stepwise, any failure during the synthesis results in the wrong product. The site for each reaction must be defined so that the reaction occurs in a rapid and efficient manner. Each step in the process should provide for a reproducible result and not interfere with the next stage or the reaction at a different site.
Since the arrays provide for a large number of different compounds, the process requires many steps. With oligonucleotides, an in situ synthesis is employed wherein each monomer addition involves a plurality of steps, so that the synthesis at each site involves the number of steps for each addition multiplied by the number of monomers in the oligonucleotide. In order to be able to produce arrays of oligonucleotides efficiently, automated systems are preferred to provide for the accurate placement of reagents, efficient reaction, close packing of different compounds and the indexing of individual oligonucleotides with a particular site in the array.
As might be imagined, relatively large chambers are required to surround the aforementioned members of the overall apparatus. Larger chambers require proportionately greater amounts of gas to control atmospheric conditions such as humidity. The flow of gas inside a large volume chamber with equipment inside is inherently difficult to control. There is the potential for stagnation zones and consequent non-uniform atmospheric conditions in side the chamber. In order to change rapidly the humidity, for example, in a large volume chamber, high flow rates of dry gas such as nitrogen are required. Furthermore, the substrate must be held at a predetermined distance from the dispensers of the dispensing apparatus in order to achieve accuracy of the dispensing process. Accordingly, configurations that have moving stages outside of the chamber and the substrate inside the chamber increase the inherent errors in the process.
There is, therefore, a need for a chamber that is relatively small in size and that allows for substantial control of the environment within the chamber. The chamber should allow devices such as dispensing elements to be incorporated in the chamber.
One embodiment of the present invention is an apparatus for preparing an array of chemical compounds on the surface of a support. The apparatus comprises two elements that are in a sealed, movable relationship relative to one another and that form a chamber having a controllable interior environment for preparing said array of chemical compounds.
Another embodiment of the present invention is an apparatus comprising a top element, a bottom element, and a mechanism for introducing a gas to form a movable aerodynamic seal between the top element and the bottom element. In this manner a chamber having a controllable interior environment is formed. The top element may have at least a portion of a device for dispensing reagents sealingly affixed therein. The bottom element may be adapted for introduction of a support into the interior of the chamber formed by the top and the bottom elements.
Another embodiment of the present invention is an apparatus for manufacturing a plurality of biopolymers on a support. A top element of the apparatus has sealingly affixed therein at least a portion of a device for dispensing reagents for synthesizing the biopolymers on a surface of the support. A bottom element of the apparatus is adapted for introduction of a support therethrough. The surface of the support comprises discrete sites that are activated for reaction with the reagents. The apparatus further comprises a mechanism for introducing a gas into a gap between the top and the bottom element to form a movable aerodynamic seal between the top element and the bottom element. In this manner a chamber is formed having a relatively small volume and a controllable interior environment. The apparatus also comprises a platform to which the support is releasably attached. The platform is adapted to move the surface of the support relative to the dispensing device and the bottom element of the apparatus relative to the top element thereof.
Another embodiment of the present invention is a method for forming a chamber having a controllable interior environment. A separate top element and a separate bottom element are disposed relative to one another to form a gap between the two elements. A gas is introduced into the gap. The pressure of the gas is sufficient to form a movable aerodynamic seal between the top element and the bottom element thereby forming the chamber. The top element may have sealingly affixed therein at least a portion of a device for dispensing reagents. The bottom element may be adapted for sealing introduction of a support therethrough.
Another embodiment of the present invention is a method for synthesizing a plurality of biopolymers on a support. A reaction chamber is formed by disposing two elements relative to one another to form a gap between the two elements. A gas is introduced into the gap to form a movable aerodynamic seal between the elements thereby forming the chamber. The support is introduced into the reaction chamber. The support and a dispensing system for dispensing reagents for the synthesis of the biopolymers are brought into a dispensing position relative to activated discrete sites on a surface of the support by moving the support and one of the elements relative to the other element. Reagents are dispensed to the discrete sites and the support and/or the dispensing system are removed from the relative dispensing position. The steps are optionally repeated until the biopolymer is formed.