Abstract:
During the light illumination period of a monomer addition cycle in synthesizing an DNA microarray, undesirable reflections of illumination light from various interfaces that the illumination light passes through near the synthesis surface of the substrate may reduce the light-dark contrast, and negatively affect the precision and resolution of the microarray synthesis. The present invention provides an flow cell that reduces the undesired reflections by constructing certain flow cell structures with materials that have similar refractive indexes as that of the solution that is in the oligomer synthesis chamber during the illumination period and/or constructing certain flow cell structures or covering the structures with a layer of a material that has a high extinction coefficient.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
         [0001]    Not applicable.  
         STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT  
         [0002]    Not applicable.  
         BACKGROUND OF THE INVENTION  
         [0003]    The sequencing of deoxyribonucleic acid (DNA) is a fundamental tool of modem biology and is conventionally carried out in various ways, commonly by processes which separate DNA segments by electrophoresis. See, e.g., “DNA Sequencing,”  Current Protocols In Molecular Biology , Vol. 1, Chapter 7 (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 are 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.  
           [0004]    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 , 91: 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 , 93:13555-13560 (November 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  119:22:5081-5090 (1997).  
           [0005]    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.  
           [0006]    A similar problem exists for synthesis of diverse sequences of other types of oligomers such as polypeptides, which is useful for determining binding affinity in screening studies. For example, Pirrung et al., U.S. Pat. No. 5,143,854 (see also PCT Application No. WO 90/15070) discloses methods of forming vast arrays of peptides using light-directed synthesis techniques. However, the large number of lithographic masks needed in the synthesis makes the fixed cost for this process relatively high and the processing time lengthy.  
           [0007]    A patterning process described in Cerrina et al., PCT Application No. WO 99/42813 overcomes the above problems. With this patterning process, an image is projected onto an activate surface of a substrate for oligomer synthesis utilizing an image former that includes a light source that provides light to a micromirror device including an array of electronically addressable micromirrors. The substrate is activated in a defined pattern and monomers are coupled to the activated sites, with further repeats until the elements of a two-dimensional array on the substrate have an appropriate monomer bound thereto. The micromirror arrays can be controlled in conjunction with an oligomer synthesizer to control the sequencing of images presented by the micromirror array in coordination with the reagents provided to the substrate. The patterning process eliminated the requirement of lithographic masks for selectively illuminating certain oligomer synthesis positions.  
           [0008]    In an instrument for the synthesis of nucleic acid probes using light, strict control of the light in the instrument proves to be a critical parameter. Light which is misdirected, inadvertently reflected or otherwise directly randomly inside the instrument, here referred to as “stray light,” can adversely affect the overall accuracy and fidelity of the arrays made by the instrument. Excess stray light can lead to the de-protection of areas of the array other than the ones intended to be de-protected, and thus cause errors in the synthesis of probes. This problem cannot be well controlled in a photolithographic process, where the use of masks interposed between the light source and the array synthesis cell inherently causes refracted light in some amount to be direct where it is not intended. However, the development of the maskless array synthesizer permits the level of stray light in the instrument to be minimized in a way that was not possible before.  
         BRIEF SUMMARY OF THE INVENTION  
         [0009]    In general, the invention is summaries in a flow cell for a microarray synthesis instrument which has a substrate onto which nucleic acid probes are to be synthesized and a block located behind the substrate, the block having a void formed in its front surface so that a flow cell is defined between the block and the substrate, the material of the block and the medium in the flow cell are selected to have substantially the same index of refraction so as tot limit stray light in the flow cell.  
           [0010]    The present invention has the advantage in that it minimizes reflected light and therefore undesired reactions during the synthesis of microarrays.  
           [0011]    It is a feature of the present invention that the utilization of light in the maskless array synthesis instrument is made more efficient.  
           [0012]    Further objects, features and advantages of the invention will be apparent from the following detailed description when taken in conjunction with the accompanying drawings.  
       
    
    
     BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS  
       [0013]    [0013]FIGS. 1 and 2 are exploded and assembled perspective views of a flow cell embodiment of the present invention for use within the instrument of FIGS. 18 and 19.  
         [0014]    [0014]FIGS. 3, 4 and  5  are top, front and side views of the assembled flow cell embodiment in FIG. 2.  
         [0015]    [0015]FIG. 6 is a perspective view of the block  13  in FIG. 1.  
         [0016]    [0016]FIGS. 7, 8 and  9  are top, bottom and side views of the block  13  in FIG. 6.  
         [0017]    [0017]FIG. 10 is a cross section view of a hole  23  of the block  13  depicted in FIG. 1, with a fluid fitting fitted in the hole.  
         [0018]    [0018]FIGS. 11, 12,  13  and  14  are exploded and assembled views of another flow cell embodiment of the present invention.  
         [0019]    [0019]FIG. 15 is a front view of the assembled flow cell embodiment in FIG. 13.  
         [0020]    [0020]FIG. 16 is a top plan view of still another flow cell embodiment of the present invention.  
         [0021]    [0021]FIG. 17 is a cross section view through the flow cell of FIG. 16 taken generally along the lines  8 - 8  of FIG. 16.  
         [0022]    [0022]FIG. 18 is a schematic view of an array synthesizer apparatus in accordance with the present invention.  
         [0023]    [0023]FIG. 19 is a schematic view of the flow cell for the instrument of FIG. 18. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0024]    The present invention is an improvement to the type of maskless microarray synthesizer described in the above-mentioned PCT Patent Application No. 99/42813, the disclosure of which is hereby incorporated by reference.  
         [0025]    In making a maskless array synthesizer, deposition of nucleic acids is determined by light deprotection of areas of the array. Since the application of light energy determines where the nucleic acids are deposited in the array, the precise control of light is a critical parameter in the quality of the array made. In fact, in making instruments intended to produce high quality arrays with optimal sequence uniformity and consistence in the DNA probes, the control of “stray light” has been found to be among the most important parameters. Stray light, as used here, refers to light which is incident onto areas of the array where is it not desired. Said in other words, stray light is light incident on a cell of the array which is supposed to be unlit at a particular time. Such stray light can lead to the addition of a nucleotide to a probe in a cell where it is not intended to add a nucleotide, thus causing sequence error in probe synthesis.  
         [0026]    There are multiple possible sources of stray light. It has been found, for example, that a reflective optical system produces less stray light delivered to the array than a refractive optical system, since in a reflective system the errant light is not focused back toward the array. It has also been found, and will be discussed in greater detail here, that reflected or refracted light in and around the reaction chamber in which the microarray is constructed can be a significant source of stray light. As will be discussed further below, the teaching of this specification are intended to illustrate techniques for and attributes of such a reaction chamber, or flow cell, can be used to minimize stray light during light-directed microarray synthesis. The result is that higher quality and more uniform microarrays can be constructed.  
         [0027]    This specification therefore describes multiple embodiments of flow cells for microarray synthesis instruments that are intended to minimize stray light creation. This is accomplished by optimizing features and parameters in the flow cell to minimize unwanted refraction or reflection of light used in the array synthesis process. The design of the flow cell can be better understood with reference to an exemplary array synthesis instrument. One exemplary instrument using a flow cell with a single reaction chamber and a optical elements light array is shown generally at  110  in FIG. 18. The apparatus includes a two-dimensional array image former  112  and a flow cell or reaction chamber  114  into which an array image is projected by the image former  112 . The flow cell, also shown in schematic fashion in FIG. 19, includes a planar substrate  116 , on the rear surface of which the microarray is synthesized. The substrate  116  is placed over a chamber  18  formed in the front of an enclosure  120 . An inlet port  122  and an outlet port  124  provide fluid communication into and out of the flow cell  114 . The image formed is constructed to direct the light pattern to the substrate  116 , where the reactions occur in the interior, or rear, surface of the substrate  116 . The areas of the substrate on which the nucleic acid probes are constructed are indicated schematically in FIG. 19 at  126 .  
         [0028]    The image former  112  allows for the direction of light from a light source  130  along an optical light path and into the flow cell reaction chamber  114  so that monomer addition reactions may occur in accordance with a pre-selected pattern. The image former  112  includes the light source  130  (e.g., an ultraviolet or near ultraviolet source such as a mercury arc lamp), an optional filter  132  to receive the output beam  134  from the source  130  and selectively pass only the desired wavelengths (e.g., the 365 nm Hg line), and a condenser lens  134  for forming a collimated beam  136 . The beam  136  is projected onto an array of optical elements  138 .  
         [0029]    The optical array  138  is preferable a two-dimensional array of small or miniature optical elements, or micromirrors, which are operable under electronic control such that they may be operated by the output of a general purpose digital computer  140  connected to the optical array  138 . The optical array  138  includes optical elements such as mirrors which are capable of, in effect, switching light in amplitude, direction, or other attribute of the light, sufficient to change a portion of the incident light from one state where that portion of the light actuates a reaction occurring in one cell on the substrate  116  in the flow cell  114 . There are several examples of optical devices which can serve as the optical array  138 . One is an array of micromirrors. Other types of suitable optical arrays include without limitation microshutters, micromirrors operated by bimorph piezoelectric actuators, and LCD shutters. The preferred embodiment is a digital light projector (DLP) integrated circuit available commercially from Texas Instruments.  
         [0030]    A micromirror array device  138  has a two-dimensional array of individual micromirrors which are each responsive to control signals supplied to the array device to tilt each individual micromirror in one of at least two directions. Control signals are provided from the computer  140  to the micromirror array device  138 . The micromirrors in the array  138  are constructed so that in a first position of the mirrors the portion of the incoming beam of light  136  that strikes an individual micromirror is deflected in a direction such that the light proceeds along the optical path toward the flow cell  114 , as described further below. In a second position of the micromirrors in the array  138 , the light from the beam  136  striking such mirrors in such second position is away from the optical path to the flow cell, with the result that this light is ultimately absorbed by the instrument without ever being incident on the flow cell  114 .  
         [0031]    The light which is directed by mirrors in the first position (i.e. toward the flow cell  14 ), is directed toward the first of two mirrors  142  and  144 , which in combination form an Offner optical system. The larger mirror  142  is concave and directs light incident onto one portion of it onto the smaller convex mirror  144 . The convex mirror  144  directs incident light to another portion of the concave mirror  142 , from which the light is directed to the flow cell  114 . The projection optics  112  serve to form an image of the pattern of the micromirror array  138  on the surface of the substrate  116 . A DNA synthesizer, indicated at  146 , is connected to supply reagents to and from the flow cell  114  through fluid piping  148  and  150 . The DNA synthesizer serve, in essence, as a source of reagents and pumping to deliver reagents to and remove solutions from the flow cell  114 .  
         [0032]    The instrument is used to construct nucleic acid probes on the substrate. In a direct photofabrication approach, the glass substrate  112  is coated with a layer of a binding layer chemical capable of binding the monomer building blocks. A photolabile protective group is adhered tot he binding layer. Light is applied by the projection system  112 , deprotecting the photolabile protective groups in defined preselected areas of the substrate  116 . The areas to be de-protected are selected by the operation of the mirrors in the micromirror array  138 , which selective direct light to or away from the substrate  116 . After the light application step, nucleotides are added to the flow cell which them chemically bond only where the de-protection of the photolabile groups has occurred (phosphoramidite DNA synthesis chemistry in the case of DNA probe synthesis). The added nucleotide also has a photolabile protective groups attached to it. This process is repeated for each of the four bases that makes up a nucleic acid monomer, and then repeated again for each level of the building probe strands in the microarray. In the end, a series of single stranded nucleic acid probes are created, the probes arranged in areas or features on the substrate. The process is simple, and if a combinatorial approach is used, the number of permutations increases exponentially. The resolution limit is presented by the linear response of the deprotection mechanism.  
         [0033]    [0033]FIGS. 18 and 19 only illustrates one embodiment of array synthesizer apparatus to which the method to correct for illumination nonuniformity disclosed by the present invention can be applied. The present invention disclosed here can also be applied to other array synthesizer apparatuses. The flow cell  114  in FIG. 19 is intended to be illustrated in schematic fashion only. The description that follows describes the preferred physical details of the actual flow cells as used in embodiments of the actual instrument.  
         [0034]    The first exemplary flow cell, shown in FIG. 1 includes a base  10 , a glass microscope slide  11 , a Kal RezTM gasket  12 , a block  13 , two fluid fittings  14 , and a screw press  17 . The slide  11  serves as the substrate for microarray synthesis. As illustrated in FIGS.  2 - 5 , the flow cell is held together by bolts  18  and  19  of a screw press  17 . On the surface  22  of the block  13 , there is a groove  29  (FIGS. 6 and 7) that is constructed to cooperate with the gasket  12 . The depth of the groove  29  is less than the thickness of the gasket  12 . When the flow cell is held together, the microarray synthesis surface  21  of the slide  11 , the gasket  12  and the void formed in the surface  22  of the block  13  together form a sealed reaction chamber or flow chamber, in which the microarray synthesis can occur. The block  13  has two holes  23  which allow fluid delivery into and out of the reaction chamber through fluid fittings  14 . The shape and positions of the holes  23  in the block  13  in relation to the gasket  12  are illustrated in FIGS.  6 - 9 . The bottom surface  25  of the holes  23  (FIG. 10) must be flat and smooth enough to accept face seal. The maximum bottom surface tilt  27  of the holes  23  (FIG. 10) is  150 . O-ring face seal  28  (FIG. 10) is used at the bottom  25  of the holes  23  for sealing purpose.  
         [0035]    The slide  11  (FIG. 1) is made of a material selected for optimization of transmission of the illumination light used for protection group de-protection and resistance to chemicals that come in contact with the slide during oligomer array synthesis. For example, when synthesizing DNA probes with NPOC as the protection group, the optimization is for 365 nm UV transmission and resistance to acids and bases and alkalis. High quality glassine slides of fused quartz are preferred. Other suitable materials for the slide, or substrate, include borosilicate glass and fused silica.  
         [0036]    During the light illumination period of an addition cycle in microarray synthesis, deprotecting light  31  is incident the oligomer synthesis surface  21  of the slide  11  (FIG. 1) through the opening  32  of the base  10  and the slide  11 . The light  31  then passes through the reaction chamber and reaches the surface  22  of the block  13 . During this light illumination period, the reaction chamber is filled with a reaction medium fluid which is matched in refractive index to the material of the substrate or slide  11 . One preferred medium is dimethyl sulfoxide (DMSO) with 1% imidazole. Water must be excluded from the flow cell during microarray synthesis using phosphoramidite chemistry to avoid excess protons being present. To reduce the reflection of the illumination light  31  at the interface of the reaction medium and the block  13 , the block  13  is constructed with a material that has a similar refractive index to that of the reaction medium, i.e. fused quartz, which has an index of refraction of 1.474 for light at a wavelength of 365 nm. For example, in the case of DNA probe synthesis, the reaction medium used in the reaction chamber or flow cell during the illumination period is usually DMSO with 1% imidazole, which has a refractive index of 1.4, matching the fused quartz. Thus the use of quartz to construct the block  13  and the DMSO/imidazole reaction medium provides matching indexes of refraction thereby ensuring that reflections at the interface between the medium and the block  13  are inherently minimized, thus eliminating one source of stray light. Other materials otherwise suitable for the block  13  can be used to make the block  13  if the refractive index is compatible at a practical level with the index of refraction of the reaction medium used.  
         [0037]    The surface  35  of the block  13  is covered with a layer of material that is selected to minimize reflection of incident light. In fact, the material selected can be any that has anti-reflective properties of light at 365 nm. This anti-reflective coating is intended to make sure that light is not reflected back as it exits the block. Behind the block can be any dark, light absorbing material or light trap, so that light does not return once it has exited the block  13 .  
         [0038]    FIGS.  11 - 15  show a second embodiment of the present invention. The slide  41  which serves as the substrate for oligomer array synthesis, Kal RezTM gasket  42  and the block  43  (FIG. 11) are the same as their counterparts in the embodiment shown in FIG. 1. The only difference between embodiment  2  and embodiment  1  is the flow cell assembly structures that secure the flow cell together. In embodiment  2 , a front plate  44  (FIG. 11) and a base  49  (FIG. 12) replace the base  10  (FIG. 1) of embodiment  1 . A back press block  45  (FIG. 11) replaces the screw press  17  (FIG. 1) of embodiment  1 . Instead of using bolts  18  and  19  (FIG. 1) to secure the flow cell together, embodiment  2  uses locating pins  46  (FIG. 11) to secure the flow cell together. In all other aspects, including the measures taken to reduce the undesired reflections of illumination light, embodiment  2  is identical to embodiment  1 .  
         [0039]    In another embodiment, shown in FIGS. 16 and 17, the flow cell in FIGS. 16 and 17 includes an aluminum housing  70 , held together by bolts  71 , having an inlet  73  connected to an input port line  20  and an outlet  75  converted to an out port line  21 . As illustrated in the cross-sectioned view of FIG. 17, the housing  70  includes a lower base  78  and an upper section  79  which are secured together over the substrate with the bolts  71 . The slide  61 , which provides the oligomer synthesis surface  62 , is held between the lower base  78  and a cylindrical gasket  81  (e.g., formed of Kal RezTM), which in turn is held into place by the upper section  79  of the housing  70 . The upper section  79  of the housing  70  has two slots  64  to hold a chamber cover  63 , which tightly fits into the slots  64 . The slide  61 , the gasket  81 , the upper section  79  of the housing  70  and the chamber cover  63  form a sealed chamber  88  for oligomer synthesis. The upper section  79  of the housing  70  has an inlet channel  85  extending from the inlet  73  to a sealed reaction chamber  88  and an outlet channel  89  extending from the reaction chamber  88  to the outlet  75 . The bolts  71  can be screwed and unscrewed to detachably secure the slide  61 . Preferably, as shown in FIG. 17, a rubber gasket  90  is mounted at the top of the base  78  to engage against the slide at a peripheral region to apply pressure to the slide against the gasket  81 .  
         [0040]    The slide  61  (FIG. 17) is made of a material selected for optimization of transmission of the illumination light used for protection group depotection and resistance to chemicals that come in contact with the slide during oligomer array synthesis. For example, when synthesizing DNA probes, the optimization is for 365 nm UV transmission and resistance to acids and bases.  
         [0041]    During the light illumination period of a monomer addition cycle, the illumination light  69  (FIG. 17) passes the chamber cover  63  and the reaction chamber  88 , and is incident upon the oligomer synthesis surface  62  of the slide  63 . During the illumination period, the reaction chamber of the flow cell is filled with the reaction medium. To reduce the reflection of the illumination light  69  at the interface of the reaction medium and the slide  61 , the slide is constructed with a material that has a similar refractive index to that of the reaction medium fluid. For example, in the case of DNA probe synthesis, the reaction medium used in the reaction chamber during the illumination period is usually DMSO with 1% imidazole, which has a refractive index of 1.47. Again, fused quartz glass has a refractive index of 1.474, which is similar to the refractive index of the reaction medium, and can be used to construct the slide  61 . Other materials otherwise suitable for the slide  61  can be used to make slide  61  as long as it is matched to the refractive index of the medium used. The surface  65  of the slide  61  is also covered with a layer of anti-reflective material, to reduce the illumination light reflection at the interface of the slide  61  and the air that fills the space  67 , or at the interface of the slide  61  and the base  78  when the rubber gasket  90  is not used.  
         [0042]    It is understood that the particular embodiments for correction for illumination nonuniformity set forth herein are illustrative and not intended to confine the invention, but embraces all such modified forms thereof as come within the scope of the following claims.