(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY(PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`14 August 2003 (14.08.2003)
`
`
`
`PCT
`
`(10) International Publication Number
`WO 03/066212 A2
`
`(51) International Patent Classification’:
`
`BO1J 19/00
`
`(81) Designated States (national): AE, AG, AL, AM, AT, AU,
`AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
`
`(21) International Application Number:©PCT/US03/02641 CZ, DE, DK, DM,DZ, EC, EE, ES, FI, GB, GD, GE, GH,
`GM, HR, HU,ID,IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC,
`LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, Mw,
`MX, MZ, NO, NZ, OM, PH, PL, PT, RO, RU, SC, SD, SE,
`SG, SK, SL, TJ, TM, TN, TR, TT, TZ, UA, UG, UZ, VC,
`VN, YU, ZA, ZM, ZW.
`
`(22)
`
`International Filing Date: 28 January 2003 (28.01.2003)
`
`(25)
`
`Filing Language:
`
`English
`
`(26)
`
`Publication Language:
`
`English
`
`(84)
`
`(30)
`
`Priority Data:
`10/062,918
`
`1 February 2002 (01.02.2002)
`
`US
`
`(71)
`
`Applicant: NIMBLEGEN SYSTEMSLLC [US/US]; 1
`Science Court, Madison, WI 53711 (US).
`
`(72)
`
`2017 Frazer Place,
`GREEN, Roland;
`Inventors;
`Madison, WI 53713 (US). PITAS, Alan;
`120 West
`Church Street, Evansville, WI 53536 (US). CERRINA,
`Francesco; 2122 Van Hise Avenue, Madison, WI 53705
`(US).
`
`Designated States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW),
`Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European patent (AT, BE, BG, CH, CY, CZ, DE, DK, EE,
`ES, FI, FR, GB, GR, HU,IE, IT, LU, MC, NL, PT, SE,SI,
`SK, TR), OAPI patent (BF, BJ, CF, CG, CI, CM, GA, GN,
`GQ, GW, ML, MR, NE, SN, TD, TG).
`
`Published:
`without international search report and to be republished
`upon receipt of that report
`
`(74)
`
`Agent: SEAY, Nicholas, J.; Quarles & Brady LLP, P.O.
`Box 2113, Madison, WI 53701-2113 (US).
`
`For two-letter codes and other abbreviations, refer to the “Guid-
`ance Notes on Codes andAbbreviations" appearing at the begin-
`ning ofeach regular issue ofthe PCT Gazette.
`
`O03/066212A2
`
`(54) Title: MICROARRAY SYNTHESIS INSTRUMENT AND METHOD
`
`(57) Abstract: During the light illumination period of a monomeraddition cycle in synthesizing an DNA microarray, undesirable
`reflections of illumination light from variousinterfacesthat the illumination light passes through near the synthesis surface of the sub-
`strate may reducethe 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.
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`PCT/US03/02641
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`MICROARRAY SYNTHESIS INSTRUMENT AND METHOD
`
`CROSS-REFERENCE TO RELATED APPLICATIONS
`
`[0001]
`
`Not applicable.
`
`STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
`
`OR DEVELOPMENT
`
`{0002}
`
`Not applicable.
`
`BACKGROUND OF THE INVENTION
`
`The sequencing of deoxyribonucleic acid (DNA)is a fundamental tool of modern
`[0003]
`biology and is conventionally carried out in various ways, commonly by processes which
`separate DNA segments byelectrophoresis. 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 ofother
`genomesof medical and agricultural importance (e.g., human, C. elegans, Arabidopsis). In the
`medical context, it will be necessary to "re-sequence" the genomeof 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 goodtargets for new drugsoridentification of appropriate genetic alterations that may be
`effective in genetic therapy. Other applicationslie in fields such as soil ecology or pathology
`where it would be desirable to be able to isolate DNA from anysoil or tissue sample and use
`
`probes from ribosomal DNA sequencesfrom all known microbesto identify the microbes present
`in the sample.
`|
`[0004)
`The conventional sequencing of DNA using electrophoresisis typically laborious
`and time consuming. Variousalternatives to conventional DNA sequencing have been proposed.
`One suchalternative 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. Nat]. Acad. Sci. USA, 91: 5022-5026 (May
`1994). In this approach, the surface of a solid support modified with photolabile protecting
`groupsis illuminated through a photolithographic mask, yielding reactive hydroxy! groupsin the
`
`-1-
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`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 probeat 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 approachesis 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 masksthat are required, and the multiple
`processing steps required for repositioning ofthe masks for every exposure, contribute to
`relatively high costs and lengthy processing times.
`
`[0006]
`
`A similar problem exists for synthesis of diverse sequencesof other types of
`
`oligomers such as polypeptides, which is useful for determining bindingaffinity 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 numberof 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 Cerrinaet al., PCT Application No. WO
`
`99/42813 overcomesthe above problems. With this patterning process, an image is projected
`
`onto an activate surface of a substrate for oligomer synthesis utilizing an image formerthat
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`includesa 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
`
`monomersare coupledto the activated sites, with further repeats until the elements of a two-
`
`dimensional array on the substrate have an appropriate monomerbound thereto. The
`
`micromirror arrays can be controlled in conjunction with an oligomer synthesizer to control the
`sequencing of images presented by the micromirrorarray in coordination with the reagents
`provided to the substrate. The patterning process eliminated the requirementoflithographic
`
`masksfor selectively illuminating certain oligomer synthesis positions.
`
`[0008]
`
`In an instrument for the synthesis of nucleic acid probes usinglight, strict control
`
`of the light in the instrumentprovesto 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 andfidelity 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
`
`betweenthe 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 permitsthe level of stray light in the instrument to be minimized in a way that was
`
`not possible before.
`
`BRIEF SUMMARYOF THE INVENTION
`
`In general, the invention is summariesin a flow cell for a microarray synthesis
`[0009]
`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 formedinits 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 ofrefraction so as tot
`limit stray light in the flow cell.
`,
`[00010]
`The present invention has the advantage in that it minimizes reflected light and
`
`therefore undesired reactions during the synthesis of microarrays.
`[00011]
`It is a feature of the present invention that the utilization of light in the maskless
`array synthesis instrument is made moreefficient.
`[00012]
`Further objects, features and advantages of the invention will be apparent from the
`following detailed description when taken in conjunction with the accompanying drawings.
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`BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
`
`[00013]
`
`Figs. 1 and 2 are exploded and assembled perspective viewsof a flow cell
`
`embodimentof the present invention for use within the instrumentof Figs. 18 and 19.
`
`[00014]
`
`Figs. 3, 4 and 5 are top, front and side viewsof the assembledflow cell
`
`embodimentin Fig.2.
`.
`[00015]
`Fig. 6 is a perspective view ofthe block 13 in Fig.1.
`[00016]
`Figs. 7, 8 and 9 are top, bottom and side viewsof the block 13 in Fig.6.
`[00017]
`Fig. 10 is a cross section view ofa hole 23 ofthe block 13 depicted in Fig. 1, with
`‘a fluid fitting fitted in the hole.
`
`[00018]
`
`Figs. 11, 12, 13 and 14 are exploded and assembled viewsof anotherflow cell
`
`embodimentofthe present invention.
`
`[00019]
`
`[00020]
`
`invention.
`
`Fig. 15 is a front view of the assembled flow cell embodimentin Fig. 13.
`
`Fig. 16 is a top plan view ofstill another flow cell embodimentof the present
`
`[00021]
`
`Fig. 17 is across section view throughthe flow cell of Fig. 16 taken generally
`
`along the lines 8-8 of Fig. 16.
`(00022)
`Fig. 18 is a schematic view of an array synthesizer apparatus in accordance with
`
`the present invention.
`
`—
`
`[00023]
`
`Fig. 19 is a schematic view ofthe flow cell for the instrumentof Fig. 18.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`Thepresent invention is an improvementto the type of maskless microarray
`[00024]
`synthesizer described in the above-mentioned PCT Patent Application No. 99/42813, the
`
`disclosure of which is hereby incorporated by reference.
`
`In making a maskless array synthesizer, deposition of nucleic acids is determined
`[00025]
`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 controlof light is a critical
`‘parameter in the quality ofthe array made. In fact, in making instruments intended to produce
`high quality arrays with optimal sequence uniformity and consistence in the DNAprobes, 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 whereis it not desired. Said
`
`in other words,stray light is light incident on a cell ofthe array which is supposedto be unlit at a
`
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`particular time. Suchstray 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.
`
`[00026]
`
`There are multiple possible sourcesof stray light. It has been found, for example,
`
`that a reflective optical system producesless stray light delivered to the array than a refractive
`
`optical system,since in a reflective system the errant light is not focused back towardthe array.
`
`It has also been found, andwill be discussed in greater detail here, that reflected or refracted light
`
`in and around the reaction chamberin which the microarray is constructed can be a significant
`
`source ofstray light. As will be discussed further below,the teaching ofthis specification are
`
`intendedto illustrate techniques for and attributes of such a reaction chamber,or flow cell, can be
`
`used to minimizestray light during light-directed microarray synthesis. The result is that higher
`
`quality and more uniform microarrays can be constructed.
`[00027]
`This specification therefore describes multiple embodimentsofflow cells for
`microarray synthesis instruments that are intended to minimize stray light creation. This is
`
`accomplished by optimizing features and parametersin 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 imageis 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 formedin the front of an enclosure 120. An inlet port 122 and
`
`an outlet port 124 provide fluid communication into and outof the flow cell 114. The image
`formedis 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 probesare constructed are indicated schematically in Fig. 19 at 126.
`
`The image former 112 allowsfor the direction of light from a light source 130
`[000238]
`along an optical light path and into the flow cell reaction chamber 114 so that monomeraddition
`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 andselectively pass only
`the desired wavelengths(e.g., the 365 nm Hgline), and a condenser lens 134 for forming a
`collimated beam 136. The beam 136 is projected onto an array of optical elements 138.
`
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`The optical array 138 is preferable a two-dimensionalarray of small or miniature
`[00029]
`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 connectedto the optical
`array 138. The optical array 138 includes optical elements such as mirrors which are capableof,
`in effect, switching light in amplitude, direction, or other attribute of the light, sufficient to
`changea portionofthe 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 ofoptical devices which can serveas the optical array 138. Oneis an array of
`micromirrors. Other types ofsuitable optical arrays include without limitation microshutters,
`micromirrors operated by bimorph piezoelectric actuators, and LCD shutters. The preferred
`embodimentis a digital light projector (DLP) integrated circuit available commercially from
`
`Texas Instruments.
`
`A micromirror array device 138 has a two-dimensionalarray of individual
`[00030]
`micromirrors which are each responsiveto control signals supplied to the array device to tilt each
`individual micromirrorin one ofat least two directions. Control signals are provided from the
`
`computer 140 to the micromirror array device 138. The micromirrorsin the array 138 are
`constructed so that in a first position of the mirrorsthe portion of the incoming beam oflight 136
`that strikes an individual micromirror is deflected in a direction such that the light proceeds along
`
`the optical path towardthe flow cell 114, as described further below. In a second position of the
`micromirrorsin the array 138, the light from the beam 136striking 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.
`[00031]
`The light which is directed by mirrorsin 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. Thelarger mirror 142 is concave anddirects light incident onto one portion ofit
`onto the smaller convex mirror 144. The convex mirror 144directs incidentlight to another
`
`portion of the concave mirror 142, from which thelight is directed to the flow cell 114. The
`projection optics 112 serve to form an imageofthe pattern of the micromirror array 138 on the
`surface of the substrate 116. A DNAsynthesizer, indicated at 146, is connected to supply
`reagentsto 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.
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`The instrumentis used to construct nucleic acid probes on the substrate. Ina
`[00032]
`direct photofabrication approach,the glass substrate 112 is coated with a layer of a binding layer
`chemical capable of binding the monomerbuilding blocks. A photolabile protective group is
`adhered tot he binding layer. Light is applied by the projection system112, 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 micromirrorarray 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
`ofthe photolabile groups has occurred (phosphoramidite DNA synthesis chemistry in the case of
`DNAprobe synthesis). The added nucleotide also has a photolabile protective groups attached to
`it. This process is repeated for each ofthe four bases that makes up a nucleic acid monomer, and
`then repeated again for each level of the building probe strandsin the microarray. In the end, a
`series of single stranded nucleic acid probes are created, the probes arrangedin areas or features
`on the substrate. The processis 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.
`[00033]
`Figs. 18 and 19 only illustrates one embodimentof array synthesizer apparatus to
`which the methodto 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. Theflow cell 114 in Fig. 19 is intendedto beillustrated in schematic
`fashion only. The description that follows describes the preferred physical details of the actual
`flow cells as used in embodimentsofthe actual instrument.
`
`Thefirst exemplary flow cell, shown in Fig. 1 includes a base 10, a glass
`[00034]
`microscopeslide 11, a Kal RezTM gasket 12, a block 13, twofluid fittings 14, and a screw press
`17. The slide 11 serves as the substrate for microarray synthesis. Asillustrated 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 ofthe groove 29 is less than the thickness of the gasket 12. Whentheflow cell is held
`together, the microarray synthesis surface 21 of the slide 11, the gasket 12 and the void formed in
`the surface 22 ofthe block 13 together formasealed reaction chamberor flow chamber, in which
`the microarray synthesis can occur. The block 13 has two holes 23 which allow fluid delivery
`into andoutof the reaction chamberthrough 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
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`surface 25 of the holes 23 (Fig. 10) must be flat and smooth enough to accept face seal. The
`maximum bottom surfacetilt 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.
`[00035]
`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 comein contact with the slide during oligomerarray synthesis. For example,
`
`whensynthesizing DNA probes with NPOCas the protection group, the optimization is for 365
`nm UVtransmission andresistanceto acids and basesand alkalis. High quality glassine slides of
`
`fused quartz are preferred. Other suitable materials for the slide, or substrate, include
`
`.
`borosilicate glass and fusedsilica.
`[00036]
`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 ofthe base 10 andthe slide 11. The light 31 then passes through the reaction
`chamberand reachesthe surface 22 of the block 13. Duringthis light illumination period, the
`
`reaction chamberis filled with a reaction medium fluid which is matchedin 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 andthe block 13, the block 13
`is constructed with a material that has a similar refractive index to that ofthe reaction medium,
`i.e. fused quartz, which has an index ofrefraction of 1.474 forlight at a wavelength of 365 nm.
`For example,in the case of DNA probe synthesis, the reaction medium usedin the reaction
`chamberorflow cell duringthe 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 ofrefraction
`thereby ensuringthat reflections at the interface between the medium andthe block 13 are
`inherently minimized, thus eliminating one source ofstray light. Other materials otherwise
`suitable for the block 13 can be used to makethe block 13 ifthe refractive indexis compatible at
`a practical level with the index of refraction of the reaction medium used.
`[00037]
`The surface 35 of the block 13 is covered with a layer of material that is selected
`to minimizereflection 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 makesure that
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`light is not reflected back.as it exits the block. Behind the block can be any dark,light —
`absorbing material or lighttrap, so that light does not return once it has exited the block 13.
`[00038]
`Figs. 11-15 show a second embodimentofthe 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 sameas their counterparts in the embodiment shown in Fig. 1. The only
`difference between embodiment 2 and embodiment1 is the flow cell assembly structures that
`
`secure the flow cell together. In embodiment2, 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.
`Insteadofusing 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 ofillumination
`light, embodiment2 is identical to embodiment1.
`[00039]
`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 outport line 21. Asillustrated in the cross-
`sectioned view ofFig. 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 gasket81
`(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 twoslots 64 to hold a chambercover 63,
`whichtightly 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 chamber88 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 chamber88 to the
`
`outlet 75. The bolts 71 can be screwed and unscrewedto detachably securethe slide 61.
`Preferably, as shown in Fig. 17, a rubbergasket 90 is mounted atthe top of the base 78 to engage
`against the slide at a peripheral region to apply pressureto the slide against the gasket 81.
`[00040]
`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 comein 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.
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`Duringthe lightillumination period of a monomer addition cycle, the illumination
`{00041]
`light 69 (Fig. 17) passes the chamber cover 63 and the reaction chamber88,andis incident upon
`the oligomer synthesis surface 62 of the slide 63. During the illumination period, the reaction
`chamberofthe flow cellis filled with the reaction medium. To reducethe reflection of the
`
`illumination light 69 at the interface of the reaction medium andthe 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 usedin the reaction
`chamberduring 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, whichis
`
`similar to the refractive index of the reaction medium,and can be usedto construct the slide 61.
`
`Other materials otherwise suitable for the slide 61 can be used to makeslide 61 as longas it is
`matchedto 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 airthat fills the space 67, or at the interface of the slide 61 and
`
`the base 78 whenthe rubber gasket 90 is not used.
`[00042]
`It is understoodthat the particular embodiments for correction for illumination
`nonuniformity set forth herein are illustrative and not intended to confine the invention, but
`embracesall such modified forms thereof as come within the scope of the following claims.
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`CLAIMS
`
`WE CLAIM:
`
`1.
`
`A flowcell for synthesizing a microarray of DNA probes, comprising:
`a substrate having a surface on which probesare synthesized; and
`a block thatis located behind and adjacentto the substrate, the block
`having a void formedinits front surface forming with a rear surface of the substrate a chamberin
`which a probe synthesis reaction can occur, the block being made ofa material having a similar
`refractive index to that of the medium thatis in the reaction chamber duringan illumination
`period.
`
`Theflow cell of Claim 1, wherein the slide is made of a material selected from the
`2.
`group consisting of borosilicate glass, fused quartz and fused silica.
`
`The flow cell of Claim 1, wherein the block is made of a material selected from
`3.
`the group consisting of fused quartz, borosilicate glass and fusedsilica.
`
`The flow cell of Claim 1, further comprising a layerof an anti-reflective material
`4.
`on a surface of the block that is in the illumination light path and not in contact with the solution
`in the reaction chamberduring an illumination period.
`
`The flow cell of Claim 1, wherein the oligomer synthesis surfaceofthe slide
`5.
`forms another wall of the reaction chamber.
`
`6.
`
`A flow cell for synthesizing an array of oligomers, comprising:
`a chambercoverhavinga first surface to receive an illumination light and
`a second surface which is opposite to the first surface to formafirst wall of an oligomer
`synthesis chamber in which oligomersynthesis reactions occur;
`a medium contained in the chamber; and
`a slide made of a material having a similar refractive index to that ofthe
`medium that is in the oligomer synthesis chamberduring an illumination period, the slide having
`a surface on which oligomers are synthesized.
`
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`7.
`
`' An apparatus for synthesizing arrays of oligomers such as DNA probes and
`
`polypeptides, the apparatus comprising:
`(i) a flow cell having in which monomeraddition reactions can be conducted
`(ii) a substrate forming the front surface of the flow cell, the array being formed on the
`
`substrate;
`
`(iii) a light source providing a light beam;
`(iv) an array ofoptical elements placedto receive the light beam from the light source and
`arranged such that each element ofthe array can be positioned to direct light along an optical axis
`or to not direct light along the optical axis;
`(v) projection optics capable of receiving the light reflected from the array of optical
`elements along the optical axis and imaging the pattern of the optical elements ontothe flow cell;
`
`and
`
`(vi) the flow cell containing a medium theindex ofrefraction of which is matched to the
`index ofrefraction of the substrate so as to minimizelight refractions at the interface between the
`
`substrate and the flow cell.
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`SUBSTITUTE SHEET (RULE 26)
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