`
`(30) Priority Data:
`60/075,641
`
`23 February 1998 (23.02.98)
`
`Us
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`PCT
`International Bureau
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(51) International Patent Classification © :
`(11) International Publication Number:
`WO 99/42813
`GOIN 21/00, 1/00, 21/01, 33/53, 33/543,
`.
`io.
`26 August 1999 (26.08.99)
`CO8K 3/00, C12Q 1/68, C12M 1/34
`(43) International Publication Date:
`
`(21) International Application Number: PCT/US99/03807|(81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR,
`BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD,
`(22) International Filing Date:
`22 February 1999 (22.02.99)
`GE, GH, GM, HU, ID, IL, IN, 1S, JP, KE, KG, KP, KR,
`KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN,
`MW,MX,NO, NZ, PL, PT, RO, RU, SD, SE, SG,SI, SK,
`SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW, ARIPO
`patent (GH, GM,KE, LS, MW, SD, SZ, UG, ZW), Eurasian
`patent (AM, AZ, BY, KG, KZ, MD,RU,TJ, TM), European
`patent (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR,
`IE, IT, LU, MC, NL, PT, SE), OAPI patent (BF, BJ, CF,
`CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG).
`
`substrate (12).
`
`(71) Applicant: WISCONSIN ALUMNI RESEARCH FOUNDA-
`TION [US/US]; 614. N. WalnutStreet, P.O. Box 7365, Madi-
`son, WI 53707-7365 (US).
`
`(72) Inventors: CERRINA, Francesco; 5496 Lacy Road, Madison,|Published
`WI 53705 (US). SUSSMAN,Michael, R.; 4810 Waukesha
`With international search report.
`Street, Madison, WI 53705 (US). BLATTNER,Frederick,
`R.
`1547 Jefferson Street, Madison, WI 53711 (US).
`SINGH-GASSON, Sangeet; Apartment 2, 3107 Stevens
`Street, Madison, WI 53705 (US). GREEN, Roland; 2017
`Frazer Place, Madison, WI 53713 (US).
`
`(74) Agents: ENGSTROM, Harry, C. et al.; Foley & Lardner,
`150 East Gilman Street, P.O. Box 1497, Madison, WI
`§3701—1497 (US).
`
`(54) Title: METHOD AND APPARATUS FOR SYNTHESIS OF ARRAYS OF DNA PROBES
`
`COMPUTER
`
`CONTROLLER
`
`(57) Abstract
`
`The synthesis of arrays of DNA probe sequences, polypeptides and the like is carried out using a patterning process on an active
`surface of a substrate (12). An image is projected onto the active surface (15) utilizing an image former (11) that includes a light source
`that provides light to a micromirror device (35) comprising an array of electronically addressable micromirrors (36). The substrate (12)
`is activated in a defined pattem and bases are coupled at the activated sites, with further repeats until the elements of a two-dimensional
`array on the substrate have an appropriate base bound thereto. The micromirrorarray (35) can be controlled in conjunction with a DNA
`synthesizer to control the sequencing of images presented by the micromirrorarray (35) in coordination with the reagents provided to the
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`™T
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`R
`TT
`UA
`UG
`US
`UZ
`VN
`YU
`ZW
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`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`
`Slovenia
`SI
`Lesotho
`LS
`Albania
`Spain
`Slovakia
`SK
`LT
`Lithuania
`Finland
`Armenia
`SN
`LU
`France
`Austria
`Luxembourg
`Senegal
`Swaziland
`LV
`Latvia
`8Z
`Gabon
`Australia
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`TD
`Monaco
`Chad
`MC
`United Kingdom
`Azerbaijan
`TG
`MD
`Togo
`Republic of Moldova
`Georgia
`Bosnia and Herzegovina
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`MG
`TJ
`Ghana
`Barbados
`Tajikistan
`Madagascar
`Turkmenistan
`MK
`Guinea
`The former Yugoslav
`Belgium
`
`Greece
`Burkina Faso
`Turkey
`Republic of Macedonia
`ML
`Mali
`Trinidad and Tobago
`Hungary
`Bulgaria
`
`Ukraine
`MN
`Treland
`Benin
`Mongolia
`Mauritania
`MR
`Tsrael
`Brazil
`Uganda
`United States of America
`MW
`Malawi
`Iceland
`Belarus
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`MX
`Uzbekistan
`Mexico
`Canada
`Ttaly
`NE
`Viet Nam
`Niger
`Japan
`Central African Republic
`
`Netherlands
`NL
`Yugoslavia
`Kenya
`Congo
`Zimbabwe
`NO
`Switzerland
`Norway
`Kyrgyzstan
`New Zealand
`NZ
`Céte d'Ivoire
`Democratic People’s
`
`Poland
`PL
`Cameroon
`Republic of Korea
`PT
`China
`Portugal
`Republic of Korea
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`RO
`Romania
`Kazakstan
`Cuba
`Russian Federation
`RU
`Saint Lucia
`Czech Republic
`
`Sudan
`sD
`Liechtenstein
`Germany
`Sweden
`SE
`Sri Lanka
`Denmark
`
`Singapore
`SG
`Liberia
`Estonia
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`KZ
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`METHOD AND APPARATUS FOR SYNTHESIS OF ARRAYS OF
`DNA PROBES
`
`FIELD OF THE INVENTION
`
`This invention pertains generally to the field of biology and particularly
`to techniques and apparatusfor the analysis and sequencing of DNA and related
`
`polymers.
`
`BACKGROUND OF THE INVENTION
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`The sequencing of deoxyribonucleic acid (DNA)is a fundamental toolof
`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
`applicationslie in fields such as soil ecology or pathology whereit would be desirable
`to be able to isolate DNA from any soil or tissue sample and use probes from
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`ribosomal DNA sequences from all known microbesto identify the microbes present in
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`the sample.
`
`The conventional sequencing of DNA using electrophoresis is typically
`laborious and time consuming. Variousalternatives to conventional DNAsequencing
`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 hydroxy] 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 occursat sites
`that had been exposedto light. Following capping, and oxidation, the substrate is
`rinsed and the surfaceis 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
`repeatedto 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 semiconductorphotoresists,
`whichare 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, 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, No. 22, 1997, pp. 5081-
`
`5090...
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`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
`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.
`
`SUMMARYOF THE INVENTION
`
`In accordance with the present invention, the synthesis of arrays of DNA
`probe sequences, polypeptides, and the like is carried out rapidly andefficiently using
`patterning processes. The process may be automated and computer controlled to allow
`the fabrication of a one or two-dimensional array of probes containing probe sequences
`customized to a particular investigation. No lithographic masks are required, thus
`eliminating the significant costs and time delays associated with the production of
`lithographic masks and avoiding time-consuming manipulation and alignment of
`multiple masks during the fabrication process of the probe arrays.
`In the present invention, a substrate with an active surface to which
`DNAsynthesis linkers have been applied is used to support the probes that are to be
`fabricated. To activate the active surface of the substrate to provide thefirst level of
`bases, a high precision two-dimensional light image is projected onto the substrate,
`illuminating those pixels in the array on the substrate active surface which are to be
`activated to bind a first base. Thelight incident on the pixels in the array to which
`light is applied deprotects OH groups and makes them available for binding to bases.
`After this developmentstep, 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
`processis then repeatedto bind anotherbase to a different set of pixel locations, until
`all of the elements of the two-dimensional array on the substrate surface have an
`appropriate base boundthereto. The bases bound onthe substrate are protected, either
`with a chemical capable of binding to the bases or with a layer(s) of photoresist
`covering all of the bound bases, and a new array pattern is then projected and imaged
`onto the substrate to activate the protecting material in those pixels to which the first
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`new base is to be added. These pixels are then exposed anda solution containing the
`selected base is applied to the array so that the base binds at the exposed pixel
`locations. This process is then repeated for all of the other pixel locations in the
`second level of bases. The process as described may then be repeated for each desired
`level of bases until the entire selected two-dimensionalarray of probe sequenceshas
`
`been completed.
`The image is projected onto the substrate utilizing an image former
`having an appropriate light source that provides light to a micromirror device
`comprising a two-dimensional array of electronically addressable micromirrors, each of
`which can be selectively tilted between one ofat least two separate positions.
`In one of
`the positions of each micromirror, the light from the source incident on the
`micromirror is deflected off an optical axis and away from the substrate, and in a
`secondof the at least two positions of each micromirror,the light is reflected along the
`optical axis and toward the substrate. Projection optics receive the light reflected from
`the micromirrors and precisely image the micromirrors onto the active surface of the
`substrate. Collimating optics may be usedto collimate the light from the source into a
`beam provided directly to the micromirror array or to a beam splitter, wherein the
`beam splitter reflects a portion of the beam to the micromirror array and transmits
`reflected light from the micromirror array through the beam splitter. The light directly
`reflected from the micromirrors or transmitted through the beam splitter is directed to
`projection optics lenses which image the micromirror array onto the active surface of
`the substrate. Because the selectively addressable micromirrorsin the micromirror
`array may either fully reflect or fully deflect the light provided to them, the image of
`the micromirror array exhibits a very high contrast between the “on” and “off” pixels.
`The micromirrors may also be capable of being indexed to more than two positions, in
`which case additional optics may be provided to allow exposure of more than one
`substrate using a single micromirror array device.
`In addition, the micromirrors are
`capable of reflecting light at any wavelength without damage to them, allowing short
`wavelength light, including light in the range of ultraviolet to near ultraviolet light, to
`be utilized from the light source.
`The micromirror array is operated under control of a computer which
`provides appropriate pixel address signals to the micromirror array to cause the
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`appropriate micromirrorsto be in their “reflect” or “deflect” positions. The
`appropriate micromirror array pattern for each activation step in each level of bases to
`be addedto the probes is programmed into the computercontroller. The computer
`controller thus controls the sequencing of the images presented by the micromirror
`array in coordination with the reagents provided to the substrate.
`In one embodiment, the substrate may be transparent, allowing the
`image of the micromirrorarray to be projected through the surface of the substrate that
`is opposite to the active surface. The substrate may be mounted within a flow cell,
`with an enclosure sealing off the active surface of the array, allowing the appropriate
`reagents to be flowed through the flow cell and over the active surface of the array in
`the appropriate sequence to build up the probesin the array.
`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 DRAWINGS
`
`In the drawings:
`Fig. 1 is a schematic view of an array synthesizer apparatus in
`accordance with the present invention.
`Fig. 2 is a schematic view of another array synthesizer apparatus in
`accordance with the present invention.
`Fig. 3 is a more detailed schematic view of a general telecentric array
`synthesizer apparatus in accordance with the invention.
`Fig. 4 is an illustrative ray diagram forthe refractive optics of the
`
`apparatus of Fig. 3.
`Fig. 5 is a schematic view of a further embodiment of an array
`synthesizer apparatus in accordance with the invention in which telecentric reflective
`optics are utilized.
`Fig. 6 is an illustrative ray diagram for the reflective optics of the
`
`apparatus of Fig. 5.
`Fig. 7 is a top plan view ofa reaction chamber flow cell which may be
`utilized in the array synthesizer apparatus of the invention.
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`Fig. 8 is a cross-sectional view through the reaction chamber flow cell
`of Fig. 7 taken generally along the lines 8-8 of Fig. 7.
`Fig. 9 is an illustrative view showing the coating of a substrate with a
`
`photolabile linker molecule.
`Fig. 10 is an illustrative view showing the photo-deprotection of the
`linker molecule and the production of free OH groups.
`Fig. 11 is an illustrative view showing the coupling of markersto free
`OH groups produced by the photo-deprotection of the linker molecules.
`Fig. 12 is an illustrative view showing the coupling of DMT-nucleotide
`to free OH groups produced from photo-deprotection of the linker molecules.
`Fig. 13 is an illustrative view showing acid deprotection of DMT-
`
`nucleotides.
`
`Fig. 14 is an illustrative view showing the hybridization of poly-A probe
`labeled with fluorescein with poly-T oligonucleotide synthesized from DMT-
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`nucleotide-CEPs.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`With reference to the drawings, an exemplary apparatus that may be
`used for DNA probearray synthesis, polypeptide synthesis, and the like is shown
`generally at 10 in Fig. 1 and includes a two-dimensional array image former 11 and a
`substrate 12 onto which the array imageis projected by the image former 11. For the
`configuration shownin Fig. 1, the substrate has an exposed entrance surface 14 and an
`opposite active surface 15 on which a two-dimensionalarray of nucleotide sequence
`probes 16 are to be fabricated. For purposes of illustration, the substrate 12 is shown
`in the figure with a flow cell enclosure 18 mountedto the substrate 12 enclosing a
`volume 19 into which reagents can be provided through an input port 20 and an output
`port 21. However, the substrate 12 may be utilized in the present system with the
`active surface 15 of the substrate facing the image former 11 and enclosed within a
`reaction chamber flow cell with a transparent window to allow light to be projected
`onto the active surface. The invention may also use an opaque or poroussubstrate.
`The reagents may be providedto the ports 20 and 21 from a conventional base
`synthesizer (not shownin Fig. 1).
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`The image former 11 includesa light source 25(e.g., an ultraviolet or
`near ultraviolet source such as a mercury arc lamp), an optionalfilter 26 to receive the
`output beam 27 from the source 25 and selectively pass only the desired wavelengths
`(e.g., the 365 nm Hgline), and a condenserlens 28 for forming a collimated beam 30.
`Other devices for filtering or monochromating the sourcelight, €.g., diffraction
`gratings, dichroic mirrors, and prisms, may also be used rather than a transmission
`filter, and are generically referred to as “filters” herein. The beam 30 is projected
`onto a beam splitter 32 which reflects a portion of the beam 30 into a beam 33 whichis
`projected onto a two-dimensional micromirror array device 35. The micromirror array
`device 35 has a two-dimensional array of individual micromirrors 36 which are each
`responsive to control signals supplied to the array device 35 to tilt in one of at least two
`directions. Control signals are provided from a computer controller 38 on contro! lines
`39 to the micromirror array device 35. The micromirrors 36 are constructed so that in
`a first position of the mirrors the portion of the incoming beam oflight 33 that strikes
`an individual micromirror 36 is deflected in a direction oblique to the incoming beam
`33, as indicated by the arrows 40. Ina second position of the mirrors 36, the light
`from the beam 33 striking such mirrors in such second position is reflected back
`parallel to the beam 33, as indicated by the arrows 41. The light reflected from each of
`the mirrors 36 constitutes an individual beam 41. The multiple beams 41 are incident
`upon the beam splitter 32 and pass through the beam splitter with reduced intensity and
`are then incident upon projection optics 44 comprised of,e.g., lenses 45 and 46 and an
`adjustable iris 47. The projection optics 44 serveto form an imageof the pattern of
`the micromirror array 35, as represented by the individual beams 41 (and the dark
`areas between these beams), on the active surface 15 of the substrate 12. The outgoing
`beams 41 are directed along a main optical axis of the image former 11 that extends
`between the micromirror device and the substrate. The substrate 12 in the
`configuration shown in Fig. 1 is transparent, e.g., formed of fused silica or soda lime
`glass or quartz, so that the light projected thereon, illustratively represented by the
`lines labeled 49, passes through the substrate 12 without substantial attenuation or
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`diffusion.
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`A preferred micromirror array 35 is the Digital Micromirror Device
`(DMD)available commercially from Texas Instruments, Inc. These devices have
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`arrays of micromirrors (each of which is substantially a square with edges of 10 to 20
`uum in length) which are capable of formingpatterned beamsof light by electronically
`addressing the micromirrors in the arrays. Such DMDdevicesare typically used for
`video projection and are available in various array sizes, e.g., 640 x 800 micromirror
`elements (512,000 pixels), 640 x 480 (VGA; 307,200 pixels), 800 x 600 (SVGA;
`480,000 pixels); and 1024 x 768 (786,432 pixels). Such arrays are discussed in the
`following article and patents: Larry J. Hornbeck, “Digital Light Processing and
`MEMs: Reflecting the Digital Display Needs of the Networked Society,” SPIE/EOS
`European Symposium on Lasers, Optics, and Vision for Productivity and
`Manufacturing I, Besancon, France, June 10-14, 1996; and U.S. Patents 5,096,279,
`5,535,047, 5,583,688 and 5,600,383. The micromirrors 36 of such devices are
`capable of reflecting the light of normal usable wavelengths, including ultraviolet and
`near ultraviolet light, in an efficient manner without damage to the mirrors themselves.
`The window ofthe enclosure for the micromirror array preferably has anti-reflective
`coatings thereon optimized for the wavelengths of light being used. Utilizing
`commercially available 600 x 800 arrays of micromirrors, encoding 480,000 pixels,
`with typical micromirror device dimensions of 16 microns per mirror side and a pitch
`in the array of 17 microns, provides total micromirror array dimensions of 13,600
`microns by 10,200 microns. By using a reduction factor of 5 through the optics system
`44, a typical and readily achievable value for a lithographic lens, the dimensionsofthe
`image projected onto the substrate 12 are thus about 2,220 microns by 2040 microns,
`with a resolution of about 2 microns. Larger images can be exposed on the substrate
`12 by utilizing multiple side-by-side exposures (by either stepping the flow cell 18 or
`the image projector 11), or by using a larger micromirror array.
`It is also possible to
`do one-to-one imaging without reduction as well as enlargement of the image on the
`substrate, if desired.
`The projection optics 44 may be of standard design, since the images to
`be formed are relatively large and well away from the diffraction limit. The lenses 45
`and 46 focusthe light in the beam 41 passed through the adjustable iris 47 onto the
`active surface of the substrate. The projection optics 44 and the beam splitter 32 are
`arranged so that the light deflected by the micromirror array away from the main
`optical axis (the central axis of the projection optics 44 to which the beams 41 are
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`parallel), illustrated by the beams labeled 40 (e.g., 10° off axis) fall outside the
`entrance pupil of the projection optics 44 (typically 0.5/5 = 0.1; 10° correspondsto an
`aperture of 0.17, substantially greater than 0.1).
`Theiris 47 is used to control the
`effective numerical aperture and to ensure that unwanted light (particularly the off-axis
`beams40) are not transmitted to the substrate. Resolution of dimensions as small as
`0.5 microns are obtainable with such optics systems. For manufacturing applications,
`it is preferred that the micromirror array 35 be located at the object focal plane of a
`lithographicI-line lens optimized for 365 nm. Such lenses typically operate with a
`numerical aperture (NA)of 0.4 to 0.5, and have a large field capability
`The micromirror array device 35 may be formed with a single line of
`micromirrors (e.g., with 2,000 mirror elements in one line) which is stepped in a
`scanning system.
`In this mannerthe height of the imageis fixed by the length of the
`line of the micromirror array but the width of the image that may be projected onto the
`substrate 12 is essentially unlimited. By moving the stage 18 which carries the
`substrate 12, the mirrors can be cycled at each indexed position of the substrate to
`define the image pattern at each new line that is imaged onto the substrate active
`
`surface.
`
`Various approaches maybe utilized in the fabrication of the DNA
`probes 16 on the substrate 12, and are adaptations of microlithographic techniques. In
`a “direct photofabrication approach,” the glass substrate 12 is coated with a layer of a
`chemical capable of binding the nucleotide bases. Light is applied by the projection
`system 11, deprotecting the OH groups on the substrate and making them available for
`binding to the bases. After development, the appropriate nucleotide base is flowed
`onto the active surface of the substrate and bindsto the selected sites using normal
`phosphoramidite DNA synthesis chemistry. The process is then repeated, binding
`anotherbase to a different set of locations. The process is simple, and if a
`combinatorial approach is used the numberof permutations increases exponentially.
`The resolution limit is presented by the linear response of the deprotection mechanism.
`Because of the limitations in resolution achievable with this method, methods based on
`photoresist technology may be used instead,as described, e.g., in McGall,et al.,
`supra.
`In the indirect photofabrication approach, compatible chemistries exist with a
`two-layer resist system, wherea first layer of, €.g., polyimide acts as a protection for
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`the underlying chemistry, while the top imagingresist is an epoxy-based system. The
`imaging step is commonto both processes, with the main requirement being that the
`wavelength oflight used in the imaging process be long enough notto excite transitions
`(chemical changes) in the nucleotide bases (which are particularly sensitive at 280 nm).
`Hence, wavelengths longer than 300 nm should be used. 365 nm is the I-line of
`mercury, which is the one used most commonly in wafer lithography.
`Another form ofthe array synthesizer apparatus 10 is shown ina
`simplified schematic view in Fig. 2.
`In this arrangement, the beamsplitter 32 is not
`used, and the light source 25, optionalfilter 26, and condenser lens 28 are mounted at
`an angle to the main optical axis (e.g., at 20° to the axis) to project the beam oflight
`30 onto the array of micromirrors 36 at an angle. The micromirrors 36 are oriented to
`reflect the light 30 into off axis beams 40inafirst position of the mirrors and into
`beams 41 along the main axis in a second position of each mirror.
`In other respects,
`the array synthesizer of Fig. 2 is the sameas that of Fig. 1.
`A more detailed view of a preferred array synthesizer apparatus which
`uses the off-axis projection arrangement of Fig. 2 is shown in Fig. 3.
`In the apparatus
`of Fig. 3, the source 25 (e.g., 1,000 W Hgarc lamp, Oriel 6287, 66021), provided
`with power from a powersupply 50 (e.g., Oriel 68820), is used as the light source
`which contains the desired ultraviolet wavelengths. The filter system 26 is composed,
`for example, of a dichroic mirror (e.g., Oriel 66226) that is used to absorb infrared
`light andto selectively reflect light of wavelengths ranging from 280 to 400 nm. A
`water-cooled liquid filter (e.g., Oriel 6127) filled with deionized water is used to
`absorb any remaining infrared. A colored glass filter (Oriel 59810) or an interference
`filter (Oriel 56531) is used to select the 365 nm line of the Hg lamp 25 with a 50%
`bandwidth of either 50 nm or 10 nm,respectively. An F/1 two element fused silica
`condenser (Oriel 66024) is used as the condenser 28, and with two plano-convex lenses
`52 (Melles Griot 01LQP033 and Melles Griot 01LQP023), forms a Kohler illumination
`system. This illumination system produces a roughly collimated uniform beam 30 of
`365 nm light with a diameter just large enough to encompass the 16 mm x 12 mm
`active area of the micromirror array device 35. This beam 30 is incident onto the
`device 35 at an angle of 20° measured from the normalto the face of the device. The
`micromirror array device 35 is located approximately 700 mm away from thelast
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`filter. When the micromirrorsare in a first position, the light in the beam 30 is
`deflected downwardly and outof the system. For example,in this micromirror device
`the mirrors in their first position may be at an angle of -10° with respect to the normal
`to the plane of the micromirrors to reflect the light well away from the optical axis.
`Whena micromirror is controlled to be deflected in a secondposition, e.g., at an angle
`of +10° with respect to the normalto the plane of the micromirrors, the light reflected
`from such micromirrorsin the second position emerges perpendicularly to the plane of
`the micromirror array in the beam 41. The pattern formed by the light reflected from
`the micromirrors in their second position is then imaged onto the active surface 15 ofa
`glass substrate 12 enclosed in a flow cell 18 using a telecentric imaging system
`composed of two doublet lenses 45 and 46 and an adjustable aperture 47. Each of the
`doublet lenses 45 and 46 is composed of a pair of plano-convexlenses (e.g., Melles
`Griot 01LQP033 and 01LQP037) put together with the curved surfaces nearly
`touching. The first doublet lens is oriented so that the shorter focal length (01LQP033)
`side is towards the micromirror array device 35, and the second doubletis oriented so
`that its longer focal length (01LQPO037)side is toward the micromirror array device 35.
`Doublets composed of identical lenses may be used, in which case either side may face
`the micromirror array device. The adjustable aperture 47, also called a telecentric
`aperture, is located at the back focal plane of the first doublet.
`It is used to vary the
`angular acceptanceofthe optical system. Smaller aperture diameters correspond to
`improve contrast and resolution but with correspondingly decreased intensity in the
`image. Asillustrated in Fig. 3, a standard DNA synthesizer 55 supplied with the
`requisite chemicals can be connected by the tubes 20 and 21 to the flow cell 18 to
`provide the desired sequence of chemicals, either under independent control or under
`control of the computer 38. A typical diameter for the aperture 47 is about 30 nm. An
`illustrative ray diagram showing the paths oflight through the lenses 45 and 46 is
`shown in Fig. 4 for this type of refractive optical system. Fans of rays originating at
`the center of the object (the micromirror device face), at the edge, and at an
`intermediate location are shown. The optical system forms an inverted image of the
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`15
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`20
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`25
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`30
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`face of the micromirror array device.
`Another embodiment of the array synthesizer apparatus using reflective
`optics is shown in Fig. 5. An exemplary system utilizes a 1,000 W Hg arc lamp 25 as
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`a light source (e.g., Oriel 6287, 66021), with a filter system formed of a dichroic
`mirror (e.g., Oriel 66228) that absorbs infrared light and selectively reflects light of
`wavelengths ranging from 350 to 450 nm. An F/1 two elementfused silica condenser
`jens (Oriel 66024) is used to produce a roughly collimated beam oflight 30 containing
`the 365 nm line but excluding undesirable wavelengths around and below 300 nm. A
`Kohler illumination system may optionally also be used in the apparatus of Fig. 5 to
`increase uniformity and intensity. The beam 30is incident onto the micromirror array
`device 35 which has an active area of micromirrors of about 16 mm x 12 mm and
`which is located about 210 nm from the snout of the UV source 25, with the beam 30
`striking the planar face of the micromirror device 35 at an angle of 20° with respect to
`a normal to the plane of the array. The light reflected from the micromirrors in a first
`position of the micromirrors, €.g.,
`-10° with respect to the plane of the array, is
`directed out of the system, whereas light from micromirrors that are in a second
`position, e.g., +10° with respect to the plane of the array, is directed in the beam 41
`toward a reflective telecentric imaging system composed of a concave mirror 60 and a
`convex mirror 61. Both mirrors are preferably spherical and have enhanced UV
`coating for high reflectivity. After executing reflections from the mirrors 60 and 61,
`the beam 41 may beincident upon another planar mirror 63 which deflects the beam
`toward the flow cell 18. The light reflected from the micromirrors is imaged onto the
`active surface of a glass substrate enclosed in the flow cell 18. A telecentric aperture
`(not shown in Fig. 5) may be placed in front of the convex mirror. The beam 41 first
`strikes the concave mirror, then the convex mirror, and then the concave mirror again,
`with the planar mirror 63 optionally being used to deflect the light 90° to direct i