(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`I lllll llllllll II llllll lllll llll I II Ill lllll lllll 111111111111111111111111111111111
`
`(43) International Publication Date
`8 February 2001 (08.02.2001)
`
`PCT
`
`(10) International Publication Number
`WO 01/08800 Al
`
`(51) International Patent Classification7:
`Cl2Q 1/68
`
`BOIL 7 /00 II
`
`(21) International Application Number: PCT/USOOl18885
`
`(22) International Filing Date:
`
`11 July 2000 (11.07.2000)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`60/146,712
`60/170,128
`
`30 July 1999 (30.07.1999) US
`10 December 1999 (10.12.1999) US
`
`INC.
`BIO-RAD LABORATORIES,
`(71) Applicant:
`[US/US]; 1000 Alfred Nobel Drive, Hercules, CA 94547
`(US).
`
`(72) Inventors: CHU, Daniel, Y., M.; 1331 47th Avenue, San
`Francisco, CA 94122 (US). RISING, Donald, L.; 1152
`
`Colusa Avenue, Berkeley, CA 94707 (US). CEREMONY,
`Jeff; 1374 Leafwood Court, Fairfield, CA 94585 (US).
`BALDWIN, Cliff; 204 Eagle Lane, Brentwood, CA 94513
`(US).
`
`(74) Agents: HEINES, M., Henry et al.; Townsend and
`Townsend and Crew LLP, Two Embarcadero Center, 8th
`floor, San Francisco, CA 94111-3834 (US).
`
`(81) Designated States (national): AE, AG, AL, AM, AT, AU,
`AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ,
`DE, DK, DM, DZ, 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, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM,
`TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW.
`
`(84) Designated States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), Eurasian
`patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
`
`[Continued on next page]
`
`(54) Title: TEMPERATURE CONTROL FOR MULTI-VESSEL REACTION APPARATUS
`
`r
`
`({
`
`(57) Abstract: Temperature control in
`a rectangular array of reaction vessels
`such as a thermal cycler such as is
`used for PCR procedures is achieved
`by use of a temperature block that
`is in contact with a combination of
`Peltier effect thermoelectric modules
`and wire heating elements embedded
`along the edges of the block. The
`elements can be energized in such
`a manner as to achieve a constant
`temperature throughout the array or a
`temperature gradient. Further control
`over the temperature and prevention of
`condensation in the individual reaction
`vessels is achieved by the use of a
`glass (or other transparent material)
`plate positioned above the vessels,
`with an electrically conductive coating
`on the upper surface of the glass plate
`to provide resistance heating.
`
`~-----.,:--- f 7
`
`;;;;;;;;;;;;;;;
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`;;;;;;;;;;;;;;;
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`;;;;;;;;;;;;;;; -
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`-;;;;;;;;;;;;;;; ==
`;;;;;;;;;;;;;;; -
`;;;;;;;;;;;;;;; -;;;;;;;;;;;;;;;
`;;;;;;;;;;;;;;; -;;;;;;;;;;;;;;;
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`;;;;;;;;;;;;;;;
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`;;;;;;;;;;;;;;;
`
`!!!!!!!!!!!!!!!
`
`THERMO FISHER EX. 1021
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`

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`WO 01/08800 Al
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`I lllll llllllll II llllll lllll llll I II Ill lllll lllll 111111111111111111111111111111111
`
`patent (AT, BE, CH, CY, DE, DK, ES, Fl, 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).
`
`For two-letter codes and other abbreviations, refer to the "Guid(cid:173)
`ance N ates on Codes and Abbreviations" appearing at the begin(cid:173)
`ning of each regular issue of the PCT Gazette.
`
`Published:
`-
`With international search report.
`
`THERMO FISHER EX. 1021
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`WO 01/08800
`
`PCT /US00/18885
`
`TEMPERATURE CONTROL FOR
`MULTI-VESSEL REACTION APPARATUS
`
`CROSS-REFERENCE TO RELATED APPLICATIONS
`
`This application is related to United States Provisional Patent Applications Nos.
`
`5
`
`60/146,712, filed July 30, 1999, and 60/170,128, filed December 10, 1999, and claims all
`
`benefits legally available from both. Each of these provisional patent applications is
`
`incorporated herein by reference for all purposes capable of being served thereby.
`
`BACKGROUND OF THE INVENTION
`
`Certain chemical syntheses involve the use of sequential reactions, cyclic reactions, or
`
`10 multiple reactions occurring simultaneously. Prominent examples of such syntheses are the
`
`polymerase chain reaction and the ligase chain reaction. The polymerase chain reaction
`
`(PCR), for example, entails a sequence of steps including denaturing a polynucleotide,
`
`annealing primer oligonucleotides to the denatured polynucleotide, and extension of the
`
`primers to synthesize new polynucleotide strands along the denatured strands. The success of
`
`15
`
`the procedure relies on high yield, high selectivity, and a controlled reaction rate at each
`
`stage. Yield, selectivity, and reaction rate often vary with temperature, and optimal
`
`temperatures in each case v~ry with such parameters as the length and nucleotide
`
`composition of the polynucleotide, and the choice of enzymes and other components of the
`
`reaction system. Determination of the optimal temperatures and accurate control of the
`
`20
`
`temperatures at the optimal levels are important in achieving success in these procedures.
`
`Other protocols and procedures involve multiple reactions performed simultaneously in
`individual reaction vessels all at the same temperatwe. Accuracy and control 8!e important
`in these procedures as well.
`
`Laboratory apparatus in which this kind of control is achieved is offered by numerous
`
`25
`
`suppliers. The typical apparatus includes one or more temperature-controlled blocks, each
`
`containing reaction wells in a two-dimensional array, with robotics to move samples between
`
`wells in a block or between different blocks and automated processing to control the
`
`temperature and drive the robotics. Examples are the RoboCycler 96 of Stratagene, the PTC-
`
`100 Thermal Cycler of MJ Research, the Perkin-Elmer DNA Thermal Cycler, and the DNA
`
`30
`
`Engine Thermal Cycler of MicroPROBE.
`
`THERMO FISHER EX. 1021
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`WO 01/08800
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`PCT /US00/18885
`
`Temperature control over the entire array of reaction wells in a two-dimensional array
`
`is often less than complete, and edge effects often arise, i.e., temperature differences at the
`
`outer wells due to their greater exposure to the atmosphere or to other instrument
`
`components. Also, temperature gradients along the well array, which would permit reactions
`
`5
`
`at different temperatures or different protocols to be performed simultaneously, are difficult
`
`to achieve.
`
`Also lacking from the units named above and similar units are features that permit the
`
`user to visually or optically observe the well contents during the course of the reaction and
`
`thereby achieve real-time detection of the progress of the reaction, and to enclose the wells
`
`10
`
`with lids to prevent evaporation of the reaction mixtures without experiencing condensation
`
`on the undersides of the lids.
`
`SUMMARY OF THE INVENTION
`
`The present invention resides in part in a temperature block that can establish
`
`either a temperature gradient across an array of reaction wells or a uniform temperature
`
`15
`
`throughout the array. The temperature block is useful as a component of a thermal cycler or
`
`other similar automated laboratory apparatus which also includes other components and
`
`features that participate in sample handling and the performance and control of multiple
`
`and/or sequential chemical reactions. One of the features of the block of the present
`
`invention is a unique ability to heat and otherwise control the temperature in all wells of the
`-
`array while eliminating edge-effects, i.e., temperature deviations in wells positioned either in
`
`20
`
`the center of the array or along the outer edge of the array due to differences in heat
`
`dissipation in these regions.
`
`The present invention also resides in a thermal cycler or similar multiple
`
`25
`
`reaction apparatus that includes a heated transparent lid. The lid performs several functions.
`One is to apply force to enclosures that are placed over the tops of the wells to ~ecure the
`contents of the wells from evaporation or other loss. Another is to press the wells down
`
`against the heating or cooling block positioned underneath the wells to achieve good thermal
`
`contact. This can be done without the use of oil which has been previously used for this
`
`purpose. A third function of the heated transparent lid is to permit sufficient light to pass and
`
`30
`
`thereby permit the user to directly detect the progress of the reactions that are taking place in
`
`the wells. A fourth function is to prevent condensation of vapors on the undersides of the
`
`well enclosures, the condensations otherwise tending to introduce variations in the
`
`2
`
`THERMO FISHER EX. 1021
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`
`compositions of the reaction mixtures. The vapors are generated in the wells by the
`
`components of the reaction mixtures, particularly when the wells are heated from below.
`
`These features of the heated transparent lid are particularly useful when the thermal cycler or
`
`multiple reaction apparatus is used for polymerase chain reactions. The lid is also useful for
`
`5 multiple reaction systems in general, both those in which the reactions are performed
`
`simultaneously and those in which they are performed sequentially.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 is an exploded perspective view from below of a temperature block in
`
`accordance with the present invention.
`
`10
`
`FIG. 2 is a plan view of an array of Peltier modules forming a portion of the
`
`block construction of FIG. 1.
`
`FIG. 3 is a perspective view from above of a portion of second temperature
`
`block in accordance with this invention, designed specifically for establishing and
`
`maintaining a temperature gradient.
`
`15
`
`FIG. 4 is an exploded vertical cross section of a thermal cycler incorporating a
`
`heated transparent lid in accordance with this invention.
`
`FIG. 5 is an enlarged cross section of one end of the internal components of
`
`the thermal cycler of FIG. 4.
`
`FIG. 6 is an enlarged cross section of an alternative design for a portion of the
`
`20
`
`structure shown in FIG. 5.
`
`DESCRIPTION OF THE SPECIFIC EMBODIMENTS
`
`While this invention can be implemented in a variety of structures and
`
`embodiments, certain specific embodiments are discµssed in detail herein to proyide an
`
`understanding of the invention as a whole.
`
`25
`
`In FIG. 1, a temperature block 11 in accordance with this invention is shown.
`
`The figure is a perspective view from below with the parts separated vertically, thereby
`
`rendering visible the underside of each layer of the block construction together with the front
`
`and right side edges of each layer. The reaction wells 12 form a rectangular array on the top
`
`surface of the upper layer or sample plate 13. Each well is a hollow cylindrical receptacle
`
`30
`
`open at the top. A common array of wells in thermal cyclers is one containing 96 wells ( 12 x
`
`8), although other arrays with more or fewer wells can be used, including single rows of
`
`3
`
`THERMO FISHER EX. 1021
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`
`wells. The sample plate is preferably constructed of thin but rigid, thermally conductive
`
`material.
`
`The center layer 14 of the block is an array of Peltier modules, which are
`
`electrically connected to function as heating and/or cooling elements for the sample plate 13
`
`5
`
`positioned above. These devices, whose construction and operation are well known among
`
`those skilled in the art, utilize the Peltier effect, in which an electric current is passed through
`
`the junction of two dissimilar electric conductors to result in the production or absorption of
`
`heat depending on the direction of the current through the junction. In Peltier modules,
`
`semiconductors such as bismuth telluride appropriately doped to create n-type and p-type
`
`10 materials serve as the dissimilar conductors. The semiconductors are connected through
`
`electric leads to a DC power source. Peltier modules are commercially available from many
`
`sources, one of which is Melcor Thermal Solutions, Trenton, New Jersey, USA.
`
`Peltier modules are commonly used in arrays in which they are positioned
`
`edge-to-edge to form a planar arrangement for attachment to a flat or smooth surface. In the
`
`15
`
`particular embodiment shown in the Figures, and seen more clearly in the plan view of FIG.
`
`2, six Peltier modules 21, 22, 23, 24, 25, 26 are shown, arranged in two rows of three
`
`modules each. The imposition of either a uniform temperature or a temperature gradient is
`
`governed by the manner in which current is applied to the Peltier modules and the wire
`
`heating elements that are described below. When a temperature gradient is desired, the
`
`20
`
`gradient can be along either of the two axes, i.e., from left to right or from front to rear. The
`
`electrical connections betwee_n the six modules and the wire heating elements will be selected
`
`to achieve a gradient in the desired direction.
`
`Returning to FIG. 1, the lower layer 15 of the block is a heat sink of
`
`conventional construction, utilizing an array of fins 16 to dissipate heat generated by the
`
`25
`
`Peltier modules. The removal of heat can also be enhanced by the placement of a fan 17
`
`below the fins, causing air to flow upward into the fins, as indicated by the arrow 18.
`
`Positioned between the Peltier module layer 14 and the sample plate 13 is a
`
`solid layer of thermally conductive material 19, and another such layer 20 is positioned
`
`between the Peltier module layer and the heat sink 15. These layers serve to improve the
`
`30
`
`dissipation of heat in the lateral directions. Any flat thermally conductive material can be
`
`used. An example is GRAFOIL flexible graphite sheets, available from UCAR Carbon Co.,
`
`Inc., Danbury, Connecticut, USA. Thermally conductive grease can be used in place of the
`
`thermally conductive layers.
`
`4
`
`THERMO FISHER EX. 1021
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`
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`
`Deviations from a uniform temperature or a controlled temperature gradient
`
`along the sample plate 13 occur either at the two side edges (left side 31 and right side 32) or
`
`at the front edge 33 and the rear edge (not visible), or at all of the edges, since regions along
`
`these edges have greater exposure to the atmosphere than regions toward the center of the
`
`5
`
`block. For the left and right side edges 31, 32, grooves 34, 35 are formed along the lengths of
`
`each of these two edges, and inserted in these grooves are electrical wire heating elements 36,
`
`37. The two elements are shown removed laterally from the grooves for ease of visibility. In
`
`certain embodiments of the invention, additional electrical wire heating elements reside in
`
`grooves along the front 33 and rear edges. Only the heating element 38 for the front groove
`
`10
`
`is shown. For these wire heating elements, one example of many suitable elements that can
`
`be used is nickel-chromium. The wire can be electrically insulated with conventional
`
`insulating material, such as KAPTON tape or tubing (polyimide products available from
`
`Phelps Dodge Industries, Trenton, Georgia, USA). Heat loss at the left and right edges, or
`
`the front and back edges, or all four edges, of the sample plate are thus reduced or eliminated
`
`15
`
`by use of these heating elements. When the heating block is to be maintained at a uniform
`
`temperature, the wire heating elements at the left and right edges, or all four wire heating
`
`elements, are controlled to compensate for heat loss at the edges. When a temperature
`
`gradient is to be imposed across the width of the heating block (from the left edge to the right
`
`edge), the two side wire heating elements 36, 37 are set to maintain different temperatures.
`
`20 When a temperature gradient is to be imposed in the direction from the front row of the
`
`reaction wells to the back ro'0"·(or vice versa), the front 38 and rear wire heating elements are
`
`set to maintain different temperatures. In either case, the two remaining heating elements can
`
`serve to maintain row uniformity, i.e., uniform temperatures within any single row in the
`
`direction perpendicular to the gradient.
`
`25
`
`The apparatus also contains alternative or additional means for reducing or
`
`preventing temperature deviations within rows with0ut using the front and rear heating
`
`elements. These means involve the use of blocks or inserts of material with a high coefficient
`
`of thermal conductivity, such as aluminum or copper. Four such blocks 41, 42, 43, 44 are
`
`shown in FIGS. 1and2, positioned in a row between the two rows of Peltier modules. These
`
`30
`
`thermally conductive blocks help dissipate the heat that accumulates at the center of the
`
`Peltier array and keep the inner edges of the Peltier modules out of direct contact.
`
`FIG. 1 illustrates an additional feature of this invention that is useful when a
`
`temperature gradient is imposed across the block in the left-to-right direction. This feature
`
`consists of additional grooves 45, 46 cut into the underside of the sample plate 13, running
`
`5
`
`THERMO FISHER EX. 1021
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`
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`parallel to the right and left edge grooves 31, 32. These additional grooves 45, 46 are
`
`designed to accommodate wire heating elements (not shown) similar to those 36, 37 that are
`
`inserted in the edge grooves. These additional heating elements are useful in stabilizing and
`
`controlling a temperature gradient in the left-to-right direction, and will be energized at
`
`5
`
`different levels according to the desired gradient. The number of grooves and heating
`
`elements is not critical to the invention. Greater or lesser numbers can be used, depending on
`
`the accuracy of the gradient that is sought to be achieved. For temperature gradients in the
`
`front-to-back direction, grooves can be used that are similar to those shown by running
`
`parallel to the front and back edge grooves.
`
`10
`
`FIG. 3 illustrates an alternative means of establishing a gradient in the left-to-
`
`right direction by use of wire heating elements in the same four grooves as those shown in
`
`FIG. 1. This view is a perspective view of the sample plate 13 from above rather than below.
`
`The rightmost heating element 51 carries the lowest current and thereby heats to the lowest
`
`temperature. The second heating element 52 (occupying the second groove 46 in the right-to-
`
`15
`
`left direction) carries an intermediate current to apply sufficient heat to establish a higher
`
`temperature than the first heating element 51. The third heating element 53 (occupying the
`
`third groove 45) ca:Ties a second intermediate current that is greater than that of the first
`
`intermediate current of the second heating element 52, thereby applying heat sufficient to
`
`establish a temperature that is higher than both the first and second heating elements. Both
`
`20
`
`the second and third heating elements are combined in the groove 34 along the left side edge
`
`25
`
`30
`
`to supply heat which is the additive combination of the heat supplied to the two intermediate
`
`grooves. The temperature along the sample plate will thus vary from the lowest value at the
`
`right edge to the highest value at the left edge. A corresponding arrangement can be made for
`
`gradients that are front-to-back rather than left-to-right.
`An alternative means of establishing a gradient in the front-to-back direction is
`by delivering power to the Peltier modules 21, 22, 23 in series as one circuit anQ.. to the Peltier
`
`modules 24, 25, 26 in series as another circuit. The two groups of Peltier modules are then
`
`controlled with sensors set at different temperatures. Uniformity of temperatures within each
`
`row can be maintained by the left heating element 36 and the right heating element 37.
`
`Control of the temperature, whether it be uniform or a gradient, can be
`
`achieved by electronic means in the same manner as that of thermal cyclers of the prior art,
`
`typically by the use of a microprocessor. Also as in the prior art, samples can be injected into
`
`the wells (or to sample holders inside the wells) and transferred from one well to another by
`
`robotics driven by stepper motors or other appropriate mechanisms. The robotics can
`
`6
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`likewise be controlled by a microprocessor. Further control of the temperature in the block as
`
`well as the temperature distribution can be achieved by the inclusion of temperature sensors
`
`embedded in the sample plate at various locations. Sensors of conventional construction and
`
`design can be used.
`
`5
`
`FIG. 4 illustrates additional aspects and features of the invention. The
`
`apparatus shown in this Figure is an assembly similar to that of FIG. 1 except in a vertical
`
`cross section, although still exploded, and containing certain additional components above
`
`the reaction wells. In this case, the lowest component in the assembly is a temperature block
`
`61 that is temperature-controlled or temperature-programmed with heating elements and
`
`10 microprocessors in the same manner as described above, with an array of fins 62 along the
`
`underside for heat dissipation. Peltier modules, other heating elements, and circuitry occupy
`
`intermediate layers 63, while the upper surface of the block contains an array of cylindrical
`
`wells 64 of heat-transmissive material. The component positioned above the temperature
`
`block is a tray of open-top reaction vessels 65. The outer contours of the open-top reaction
`
`15
`
`vessels 65 conform in shape to the inner profiles of the cylindrical wells 64 such that the tray
`
`can be placed over the temperature block with the reaction vessels resting inside the wells of
`
`the block in a close fit with full contact for thermal communication between the temperature
`
`block and the interiors of the reaction vessels. The contours are conical to provide access to
`
`all liquids in each reaction vessel for purposes of achieving efficient transfer of liquids and
`
`20 washing. Alternatively, the tray can be replaced by individual receptacles or by well strips
`
`each containing a row of rec~ptacles. In either case, the cylindrical wells stabilize the
`
`reaction vessels by holding them in a fixed position, while also serving as a heat transfer
`
`medium to control the temperature of the reaction vessels by either transferring heat to them
`
`or removing heat from them. Although only one row of reaction vessels is visible in the
`
`25
`
`drawing, the rectangular array may be 8 x 12 sample tubes (96 tubes total), 6 x 10 (60 total),
`
`16 x 24 (384 total), or any other number and arrangement that would be compatible with an
`
`automated system for sample manipulation and detection.
`
`Positioned above the reaction vessel tray is a sealing sheet 71 of transparent
`
`material for enclosing the tops of the reaction vessels. Positioned above the sealing sheet is a
`
`30
`
`pressure plate assembly 72 which forces the sealing sheet down over the reaction vessels.
`
`The pressure plate assembly 72 contains an apertured plate 73 at its lower extremity, each
`
`aperture 7 4 aligned with one of the reaction wells, with the circular edge of each aperture
`
`directly above the raised rim 75 at the top of each reaction vessel. The sealing sheet 71 may
`
`7
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`THERMO FISHER EX. 1021
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`be coated with a transparent adhesive to contact the raised rims 75 of the reaction vessels.
`
`The apertured plate 73 thus serves as a means for transmitting downward pressure to the
`
`reaction vessels. The apertured plate also serves two additional functions. The first is to
`
`distribute the heat generated from above (by the conductive coating on the glass plate directly
`
`5
`
`above it, as described below), thereby helping to make the heat distribution uniform. The
`
`second is to serve as an optical mask to block the passage of light from areas surrounding the
`
`reaction vessels. In systems that include automated detection methods such as those that
`
`measure fluorescent emissions from the sample tubes, the optical mask reduces noise and
`
`interference in the detected signals.
`
`10
`
`Directly above the apertured plate 73 is a transparent glass plate 76 whose
`
`upper surface 77 is coated with a thin film of electrically conductive material. The heat that
`
`is generated when electric current is passed through this film warms the sealing sheet 71 that
`
`seals the open tops of the reaction vessels and prevents condensation of vapors from the
`
`reaction mixtures on the sealing sheet. The material used as the coating 77 and the thickness
`
`15
`
`of the coating are selected to make the coating substantially transparent in addition to
`
`achieving the desired resistance. The passage of current across the plate and through the
`
`coating causes the coating to provide resistance heating, while the transparency of the plate
`
`and coating permit direct user observation or other forms of optical detection of the contents
`
`of the sample tubes from above the glass plate. Various electrically conductive coating
`
`20 materials suitable for this purpose are known to those skilled in the art, and glass plates
`
`coated with such materials ar_e available from commercial glass suppliers. Examples of
`
`suitable coating materials are tin oxide and indium/tin oxide. Glass coated with these
`
`materials may be obtained from Thin Film Devices Inc., Anaheim, California, USA, and
`
`Abrisa Industrial Glass, Ventura, California, USA. Aside from the considerations mentioned
`
`25
`
`above, the thicknesses of the glass and the coating are not critical to the invention and may
`
`vary. In most cases, the glass thickness will range frpm about 0.06 inch (0.15 cr,;n) to about
`
`0.2 inch (0.51 cm), and the coating thickness will be selected to achieve the desired
`
`resistivity. For indium/tin oxide coatings, a typical thickness may range from about 750 to
`
`about 1400 Angstroms and a typical resistivity may range from about 10 to about 50 ohms
`
`30
`
`per square. In a presently preferred embodiment, the glass is 0.09 inch (0.23 cm) in thickness
`
`and the coating resistivity is 30 ohms per square.
`
`A further component of the pressure plate assembly 72 is a pair oflenses 81,
`
`82 that direct the image or light emerging from the reaction vessels 65 and passing through
`
`the sealing sheet 71, the apertured plate 73, and the flat glass plate 76 with its conductive
`
`8
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`coating 77. In this embodiment of the invention, the lenses are angled to avoid reflection or
`
`glare.
`
`The manner in which the temperature block, reaction vessel tray, sealing sheet,
`
`and tJ1e pressure plate assembly are combined is shown in the enlarged view of FIG. 5. In
`
`5
`
`this Figure, the sealing sheet 71 is a flat sheet covering the entire reaction vessel tray 65. An
`
`alternative configuration is shown in FIG. 6, in which individual domed caps 87 with
`
`peripheral flanges 88 are used. In either case, the apertured plate is chamfered around each
`
`aperture to increase the visibility of the interiors of the reaction vessels while still pressing
`
`the sealing sheet 71 or the flanges 88 against the raised rims 75 of the reaction vessel.
`
`10
`
`The glass plate 76 whose upper surface is coated with a resistance heating film
`
`77 rests above the apertured plate, and electric current is supplied to the film by contact strips
`
`84 (only one is shown; a second strip is positioned along the opposite edge of the glass plate)
`
`that function as electric leads. The strips are conveniently bonded to the coated glass along
`
`opposite edges with electrically conductive adhesive such as silver-filled epoxy (Epoxies
`
`15
`
`Etc., Greenville, Rhode Island, USA). Alternatively, conductive metal bus bars can be
`
`applied as coatings on the conducting glass. The strips or bus bars are connected to
`
`appropriate circuitry and a power source such that power can be supplied or removed at will
`
`and controlled at variable levels.
`
`Returning to FIG. 4, the pressure plate assembly 72 is mounted in a frame 85
`
`20
`
`that holds the plate components together. Included on the frame is a protective transparent
`
`window 86. The window prevents air movement above underlying components and prevents
`
`the escape of heated air, thereby contributing to the temperature control of the system. The
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`frame also contains internal springs (not shown) which permit the frame to apply pressure to
`
`seal the reaction vessels. Positioned above the frame are conventional optical components 90
`
`25
`
`for monitoring the progress of the reactions in each reaction vessel.
`
`.
`The frame 85 is equipped for moverritfnt in the vertical direction t.o raise and
`. .
`lower the pressure plate assembly as needed, and to contact the pressure plate assembly with
`
`the temperature block 61 to form an enclosure to prevent exposure to external air
`
`disturbances.
`
`30
`
`All materials and electrical components used in this apparatus are readily
`
`available from commercial suppliers.
`
`9
`
`THERMO FISHER EX. 1021
`
`

`
`WO 01/08800
`
`WE CLAIM:
`
`PCT/US00/18885
`
`1.
`
`A temperature control block to support a plurality of reaction vessels in
`
`a rectangular arrangement while controlling the temperature of each vessel, said block
`
`compnsmg:
`
`5
`
`10
`
`a plurality of wells arranged in a rectangular array on one side of said block,
`
`each well sized to receive one such reaction vessel;
`
`at least one Peltier effect thermoelectric module affixed to said block on a side
`
`opposite that of said wells;
`
`a first wire heating element affixed to said block along one edge of said
`
`rectangular array; and
`
`a second wire heating element affixed to said block along a second edge of
`
`said rectangular array opposite said first edge.
`
`2.
`
`A temperature control block in accordance with claim 1 in which said
`
`first and second wire heating elements are independently controllable such that said elements
`
`15
`
`can be set to different temperatures, thereby forming, in conjunction with said at least one
`
`Peltier effect thermoelectric module, a temperature gradient across said rectangular array of
`
`wells, or to the same temperature, thereby forming, in conjunction with said at least one
`
`Peltier effect thermoelectric module, a uniform temperature throughout said rectangular array
`
`of wells.
`
`20
`
`3.
`
`A temperature control block in accordance with claim 1 comprising at
`
`least two Peltier effect thermoelectric modules independently controllable to be set at
`
`different temperatures, thereby forming, in conjunction with said first and second wire
`
`heating elements, a temperature gradient across said rectangular array of wells.
`
`4.
`
`A temperature control block in accordance with claim 1 ih which said
`
`25 wells have tapering profiles to conform to reaction vessels with tapering exteriors.
`
`5.
`
`A temperature control block in accordance with claim 1 further
`
`comprising cooling fins affixed to said block to dissipate heat released by said at least one
`
`Peltier effect thermoelectric module.
`
`10
`
`THERMO FISHER EX. 1021
`
`

`
`WO 01108800
`
`PCT /US00/18885
`
`6.
`
`A temperature control block in accordance with claim 1 further
`
`comprising third and fourth wire heating elements affixed to said block along edges
`
`orthogonal to those to which said first and second wire heating elements are affixed.
`
`7.
`
`Apparatus for conducting a plurality of chemical reactions
`
`5
`
`simultaneously in individual enclosed reaction vessels while monitoring the reaction in each
`
`vessel, said apparatus comprising:
`
`a plurality of open-top reaction vessels;
`
`a support block in which are formed a plurality of wells shaped to receive said
`
`open-top reaction vessels;
`
`transparent lid means for enclosing the open tops of each of said reaction
`
`vessels;
`
`a transparent plate arranged to press said transparent lid means against said
`
`open tops of said reaction vessels, said transparent plate coated with a
`
`transparent resistance heating film; and
`
`means for supplying electric current to said resistance heating film.
`
`10
`
`15
`
`8.
`
`Apparatus in accordance with claim 7 further comprising an apertured
`
`plate positioned between said transparent lid means and said transparent plate to transmit
`
`pressure from said transparent plate to said transparent lid means, the apertures of said
`
`apertured plate aligned with the wells of said support block.
`
`20
`
`9.
`
`Apparatus in accordance with claim 7 in which said transparent plate is
`
`glass and said transparent resistance heating film is a member selected from the group
`
`consisting of tin oxide and a mixture of indium and tin oxide.
`
`