(12) United States Patent
`Tretiakov et al.
`
`Illlll llllllll Ill lllll lllll lllll lllll lllll 111111111111111111111111111111111
`US006556940Bl
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 6,556,940 Bl
`Apr. 29, 2003
`
`(54) RAPID HEAT BLOCK THERMOCYCLER
`
`6,261,431 Bl * 7/2001 Mathies et al. .......... 435/286.1
`
`
`(75)
`
`Inventors: Alexandre Tretiakov, Jena (DE);
`Hans-Peter Saluz, Oberbodnitz (DE)
`
`(73) Assignee: Analytik Jena AG, Jena (DE)
`
`( *) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(21) Appl. No.:
`
`09/719,125
`
`(22) PCT Filed:
`
`Apr. 5, 2000
`
`(86) PCT No.:
`
`PCT/EP00/03224
`
`§ 371 (c)(l),
`(2), ( 4) Date: Dec. 7, 2000
`
`(87) PCT Pub. No.: W000/61797
`
`PCT Pub. Date: Oct. 19, 2000
`
`(30)
`
`Foreign Application Priority Data
`
`FOREIGN PATENT DOCUMENTS
`
`DE
`DE
`WO
`WO
`
`4022792
`19739119
`WO 98/43740
`WO 00/25920
`
`2/1992
`3/1999
`10/1998
`5/2000
`
`OTHER PUBLICATIONS
`
`Analytical Biochemistry 186, 328-331 (1990) "Minimizing
`
`the Time Required for DNAAmplification by Efficient Heat
`
`Transfer to Small Samples" by Carl T. Wittwer et al.
`
`Anal. Chem. 1998, 70, 2997-3002, "Capillary Tube Resis­
`
`tive Thermal Cycling" by Neal A Friedman, et al..
`
`The 7'h International Conference on Solid-State Sensors and
`
`Actuators, 924-926, "DNAAmplification with Microfabri­
`
`cated Reaction Chamber" by M. Allen Northrup et al.
`
`
`(List continued on next page.)
`
`Primary Examiner-Bryan Bui
`(74) Attorney, Agent, or Firm-Jordan and Hamburg LLP
`
`Apr. 8, 1999
`
`(EP) ............................................ 99106900
`
`
`(57)
`
`ABSTRACT
`
`Int. Cl.7 ............................ GOlK 5/00; C12M 1/00
`
`(51)
`(52) U.S. Cl. .................... 702/130; 702/132; 435/286.1;
`435/287.2
`(58) Field of Search ................................. 702/130, 132,
`
`702/136, 170, 97-99; 435/286.1, 286.6,
`287.2, 288.1; 422/99, 104, 600--601
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`10/1995 Wittwer et al.
`
`5,455,175 A
`5,475,610 A * 12/1995 Atwood et al. . . . . . . . . . . . . . 700/269
`
`3/1996 Pfost et al.
`
`5,496,517 A
`4/1996 Hansen et al.
`
`5,508,197 A
`10/1997 Northrup et al.
`
`5,674,742 A
`1/1998 Atwood et al.
`
`5,710,381 A
`2/1998 Baier et al.
`
`5,716,842 A
`5,721,136 A * 2/1998 Finney et al. ............ 435/287.2
`
`5,802,816 A
`9/1998 Dietzel
`
`
`A heat block thermocycler to perform rapid PCR in multiple
`small-volume samples (1-20 µl) employing, low profile, low
`thermal mass sample block the temperature of which can be
`rapidly and accurately modulated by a single thermoelectric
`pump (thermoelectric module). An array of spaced-apart
`sample wells is formed in the top surface of the block. The
`samples are placed into the wells of ultrathin-walled (20-40
`µm) multiwell plate and located into the sample block. The
`heated lid tightly seals the individual wells by pressing the
`sealing film to the top surface of the multiwell plate. Air
`pressure arising inside the tightly sealed wells at elevated
`temperatures deforms the elastic walls of the wells of the
`ultrathin-walled plate and brings them into close thermal
`contact with the sample block. A gasket thermally isolates
`the sample block from the heated lid. The PCR reactions (30
`cycles) can be performed in 10-30 minutes.
`
`21 Claims, 3 Drawing Sheets
`
`10
`
`14
`
`THERMO FISHER EX. 1041
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`

`US 6,556,940 Bl
`Page 2
`
`OIBER PUBLICATIONS
`
`Nucleic Acids Research, 1997, vol. 25, No. 15, "Optimiza­
`tion of the performance of the polymerase chain reaction in
`silicon-based microstructures" by Theresa B. Taylor et al..
`Science, vol. 280, May 15, 1998, 1046-1048, "Chemical
`Amplification: Continuous-Flow PCR on a Chip" by Martin
`U. Kopp et al..
`
`Product Application Focus, vol. 10, No. 1, (1991) 102-112,
`
`"A High-Performance System for Automation of the Poly­
`
`merase Chain Reaction" by Haff et al.
`
`
`"Rapid Thermal Cycling and PCR Kinetics" Carl T. Wittwer
`
`and Mark G. Hermann pp. 211-228, copyright 1999.
`
`Products and Applications for the Laboratory eppendorf
`
`p143, 2002.
`
`T Robot Thermocyler Whatman Biometra pp. 1-4, Jul.
`
`2001.
`
`Innovative PCR Plastics, Robbins pp. 1-10, copyright 1998.
`
`PCR Instruments and Consumables 3pgs.
`
`
`* cited by examiner
`
`THERMO FISHER EX. 1041
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`

`

`U.S. Patent
`
`Apr. 29, 2003
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`Sheet 1of3
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`US 6,556,940 Bl
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`00000
`000000
`000000
`000000
`000000
`000000
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`THERMO FISHER EX. 1041
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`

`U.S. Patent
`
`Apr. 29, 2003
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`Sheet 2 of 3
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`US 6,556,940 Bl
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`~t----11---+-~~~~
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`THERMO FISHER EX. 1041
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`

`U.S. Patent
`
`Apr. 29, 2003
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`Sheet 3 of 3
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`US 6,556,940 Bl
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`Fig. 3
`
`
`T [°C]
`
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`95
`
`
`72
`
`
`55
`
`
`o i o 20 30 40 Time [seconds]
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`THERMO FISHER EX. 1041
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`

`1
`RAPID HEAT BLOCK THERMOCYCLER
`
`US 6,556,940 Bl
`
`2
`multiwell plates. One known improvement of heat block
`temperature cycling of samples contained in plastic tubes
`has been described by Half et al. (Biotechniques, 10,
`106-112, [1991] and U.S. Pat. No. 5,475,610). They
`describe a special PCR reaction-compatible one-piece
`plastic, i.e. polypropylene, microcentrifuge tube, i.e. a thin­
`walled PCR tube. The tube has a cylindrically shaped upper
`wall section, a relatively thin (i.e. approximately 0.3 mm)
`conically- shaped lower wall section and a dome-shaped
`bottom. The samples as small as 20 µl are placed into the
`tubes, the tubes are closed by deformable, gas-tight caps and
`positioned into similarly shaped conical wells machined in
`the body of the heat block. The heated cover compresses
`each cap and forces each tube down firmly into its own well.
`The heated platen (i.e. heated lid) serves several goals by
`supplying the appropriate pressure to the caps of the tubes:
`it maintains the conically shaped walls in close thermal
`contact with the body of the block; it prevents the opening
`of the caps by increased air pressure arising in the tubes at
`elevated temperatures. In addition, it maintains the parts of
`the tubes that project above the top surface of the block at
`95°-100° C. in order to prevent water condensation and
`sample loss in the course of thermocycling. This made it
`possible to exclude the placing of mineral oil or glycerol into
`the wells of the block in order to improve the heat transfer
`to the tubes and the overlaying of the samples by mineral oil
`that prevented evaporation but also served as added thermal
`mass. In addition, the PCR tubes can be put in a two-piece
`holder (U.S. Pat. No. 5,710,381) of an 8x12, 96-well micro-
`plate format, which can be used to support the high sample
`throughput needs with any number between 1 and 96
`individual reaction tubes. When compared to conventional
`microcentrifuge tubes the use of thin-walled 0.2-ml PCR
`tubes made it possible to reduce the reaction time from 6-10
`hours to 2-4 hours or less. At the same time it was also
`shown in DE 4022792 that the use of thin-walled polycar­
`bonate microplates allows to reduce the reaction time to less
`than 4 hours. A recent improvement concerning the ramping
`rate (i.e. 3-4° C./second) of commercial thermoelectric
`(Peltier effect) heat block thermocyclers did not influence
`considerably the total reaction time. Moreover, it was con­
`cluded that a further increase in ramping rates will not be of
`a practical benefit due to the limited rate of heat transfer to
`the samples contained in thin-walled PCR tubes (see WO
`98/43740).
`
`SUMMARY OF THE INVENTION
`The present invention bears some similarity to conven­
`tional heat block thermoelectric thermocyclers for perform­
`ing PCR in plastic microplates (for example, see WO
`98/43740 and DE 4022792). However, in contrast to con­
`ventional heat block thermocylers, it provides the means for
`performing PCR, i.e. 30 cycles, in 1-20 µl samples in 10-30
`minutes. More specifically, it provides a rapid heat block
`thermocycler for convenient, high-throughput and
`inexpensive, oil-free temperature cycling of multiple small­
`volume samples.
`Accordingly, the invention concerns a heat block ther­
`mocycler for subjecting a plurality of samples to rapid
`thermal cycling, the heat block thermocycler including:
`a unit for holding a plurality of samples having
`an ultrathin-walled multiwell plate having an array of
`conically shaped wells and a low thermal mass
`sample block having an array of similarly shaped
`wells, wherein the height of the wells of the said
`multiwell plate is not more than the height of the
`wells of the said sample block,
`
`10
`
`20
`
`25
`
`30
`
`BACKGROUND OF THE INVENTION
`The invention relates to thermocyclers for an automatic
`performance of polymerase chain reaction (PCR), particu­
`larly to rapid thermocyclers. More specifically, it relates to
`rapid heat block thermocyclers for parallel processing of
`multiple small-volume samples. The present invention is
`especially useful for rapid, high-throughput, inexpensive
`and convenient PCR-based DNA-diagnostic assays.
`Since it's first published account in 1985 polymerase
`chain reaction has been transformed into myriad array of
`methods and diagnostic assays. Temperature cycling of
`samples is the central moment in PCR. In recent years 15
`various rapid thermocyclers have been developed to address
`the slow processing speed and high sample volumes of
`conventional heat block thermocyclers. These rapid ther­
`mocyclers can be divided into two broad classes:
`1. Capillary thermocyclers hold the samples within a glass
`capillary and supply heat convectively or conductively to
`the exterior of the capillary. For the description see
`Wittwer, C. T., et al., Anal.Biochem. 186: p328-331
`(1990); Friedman, N. A, Meldrum, D. R. Anal. Chem.,
`70: 2997-3002 (1998) and U.S. Pat. No. 5,455,175.
`2. Microfabricated thermocyclers are thermocyclers con­
`structed of microfabricated components; these are gener­
`ally etched structures in glass or silicon with heat supplied
`by integral resistive heating and rejected passively (or
`actively) to ambient by the structure. However, other
`schemes of thermocycling, as continuous flow thermocy­
`cling of samples are also used. For the description see
`Northrup, M. A, et al., Transducers 1993: 924--926
`(1993); Taylor, T. B., et al, Nucleic Acid Res., 25: pp
`3164--3168 (1997); Kopp, M. U. et al., Science, 280: 35
`1046-1048 (1998); U.S. Pat. No. 5,674,742; U.S. Pat. No.
`5,716,842.
`Both classes of rapid thermocyclers employ the increased
`surface-to-volume ratio of the reactors to increase the rate
`of-heat transfer to small samples (1-20 µl). Total DNA
`amplification time is reduced to 10-30 minutes. Conven­
`tional heat block thermocyclers usually take 1-3 hours to
`complete temperature cycling of 20-100 µl samples.
`However, with these benefits also several disadvantages
`appear. Increased surface area between reagents and reactors 45
`causes a loss of enzyme activity. Furthermore, DNA can also
`be irreversibly adsorbed onto silica surface of the reactors,
`especially in the presence of magnesium ions and detergents
`that are the standard components of a PCR mixture.
`Therefore, PCR in glass-silicon reactors requires the addi­
`tion of carrier protein (e.g. bovine serum albumin) and a
`rigorous optimization of the composition of the reaction
`mixture.
`Another disadvantage of these reactors is the very com­
`plicated way of loading and recovering the samples. In 55
`addition, standard pipetting equipment is usually not com­
`patible with such reactors. These inconvenient and cumber­
`some procedures are also time-consuming and labor­
`sensitive, thus limiting the throughput of the thermocyclers.
`Finally, although the reagents costs drop with a volume
`reduction to 1-10 µl, the final costs are relatively high due
`to a high cost of capillary and, especially, microfabricated
`reactors.
`Therefore, it is surprising that only little research has been
`conducted to improve the basic performance in sample size 65
`and speed of the widely used, conventional heat block
`thermocycling of samples contained in plastic tubes or
`
`40
`
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`
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`
`THERMO FISHER EX. 1041
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`US 6,556,940 Bl
`
`3
`a unit for heating and cooling the sample block compris­
`ing at least one thermoelectric module, and
`a device for sealing the plurality of samples comprising a
`high-pressure heated lid.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`The invention is more specifically illustrated by the
`accompanying figures:
`FIG. 1 illustrates a diagram of an ultrathin-walled
`microwell plate;
`FIG. 2 illustrates a diagram of a rapid heat block ther­
`mocycler; and
`FIG. 3 illustrates a chart of temperature/time profile of the
`sample block.
`
`10
`
`15
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`4
`
`plates equilibrates with the temperature of the sample block
`(4) in 1-3 seconds. For comparison, it takes 15-20 seconds
`to equilibrate the temperature of, for example a 25-µl sample
`with the temperature of the sample block when the samples
`are contained in conventional thin-walled PCR tubes. The
`other principal advantage of the use of low-profile plates
`with relatively large openings of the wells (i.e. a diameter of
`4 mm) for rapid temperature cycling of multiple samples is
`that small samples can be rapidly and accurately placed into
`the wells by means of conventional pipetting equipment. In
`this case no special skills are necessary when compared to
`the time consuming and labor-intense loading of capillaries
`or microreactors.
`The second aspect of the invention concerns the use of a
`low profile, low thermal capacity, for example the industry
`standard, silver sample blocks for holding the multiwell
`plates. A sample block (4) has a major top surface and a
`major bottom surface. An array of spaced-apart sample wells
`is formed in the top surface of the block. Usually the height
`of the block is not more than 4 mm. The thermal capacity of
`the blocks for holding 36-96-well plates is in the range of
`4.5-12 Joules/K. The blocks supply an average thermal
`mass load of 0.5--0.6 Joules/K onto 1 cm2 of the surface of
`thermoelectric module (12). Using industry standard high
`temperature, single-stage thermoelectric modules with
`maximum heat pumping power of 5-6 Watts/cm2 of the
`surface area of the module the temperature of the sample
`blocks can be changed at the ramping rate of 5-10°
`C./second (FIG. 3). Usually, single industry standard ther­
`moelectric modules, i.e. 30 mmx30 mm and 40 mmx40 mm,
`are used for temperature cycling using 36 and 64-well plates,
`respectively. A single thermoelectric module for heating and
`cooling has the advantage of an improved thermal contact
`between the module (12) and the sample block ( 4) and the
`module and an air-cooled heat sink (13) when compared to
`the use of multiple modules due to the height differences
`between the module. A thermocouple (14) with a response
`time not greater than 0.01 seconds is used for sensing the
`temperature of the sample block ( 4). The thermal mass of the
`copper heat sink (13) is usually in the range of 500--700
`Joules/K. The relatively large thermal mass of the heat sink
`(13) compared to the thermal mass of the sample block ( 4)
`compensates the increased average heat load on the heat sink
`(13) during rapid thermocycling. A programmable controller
`(10) is used for a precise time and temperature control of the
`sample block (4).
`The third aspect of the invention is, that, in order to ensure
`an efficient and reproducible sealing of small samples (5) by
`using heated-lid technology, the height of the conically
`shaped wells (2) is not greater than the height of the
`similarly shaped wells machined in the body of the sample
`block ( 4) of the thermocycler. Due to the small surface of the
`bottom of the well of the plate, their is no need of a tight
`thermal contact between the bottom of the well and the body
`of the sample block. This is in contrast to DE 4022792,
`where a precise fitting of a large spherical bottom is needed
`for an efficient heat transfer. Thus, as shown in FIG. 2, the
`geometry of the wells enables the positioning of the entire
`multiwell plate (1) into the sample block ( 4). In this case the
`pressure caused by a screw mechanism (6) of the heated lid
`is actually directed to those parts of the multiwell plate
`which are supported by the top surface of the sample block
`(4) and not to the thin walls of the wells of the plate as it is
`the case for the PCR tubes or conventional PCR plates (see
`U.S. Pat. No. 5,475,610). This advantage makes it possible
`to increase the sealing pressure of the heated lid several fold
`(i.e. 5-10 fold) compared to the conventionally used pres­
`
`20
`
`30
`
`A first aspect of the present invention concerns the use of
`low-profile, high sample density, ultrathin-walled multiwell
`plates (1) with considerably improved, i.e. 10-fold heat
`transfer to small, low thermal mass biological samples (i.e.
`1-20 µl) (5) when compared to U.S. Pat. No. 5,475,610 and
`DE 4022792. Such plates can be produced, for example, out 25
`of thin thermoplastic films by means of various thermoform­
`ing methods.
`Such thermoplastic films are, for example, polyolefin
`films, such as metallocene-catalyzed polyolefin films and/or
`copolymer films. Usually, the multiwell plate is vacuum
`formed out of cast, unoriented polypropylene film,
`polypropylene-polyethylene copolymer films or
`metallocene-catalyzed polypropylene films. The film is
`formed into a negative ("female") mould including a plu­
`rality of spaced-apart, conically shaped wells which are 35
`machined in the body of a mould in the shape of rectangular­
`or square-array. A thickness of the film for vacuum forming
`conically shaped wells is chosen according to the standard
`rule used for thermoforming, i.e. thickness of the film=well
`draw ration x thickness of the wall of the formed well.
`For example, vacuum forming wells with a draw ratio of
`two and an average thickness of the walls of 30 microns
`results in a film thickness of 60 microns. The average
`optimum wall thickness was found to be 20-40 microns. The
`draw ratio is usually in the range of 2-3. The thickness of the 45
`film is usually 50-80 microns. The thickness of a small
`dome-shaped bottom is usually 10-15 microns. Using the
`heat-transfer equation as described in DE 4022792 it can be
`shown that the rate of heat transfer is increased approxi­
`mately 10-fold when compared to U.S. Pat. No. 5,475,610
`and DE 4022792.
`A volume of the wells is usually not more than 40 µl,
`preferably 16 µl or 25 µl, a height of the wells is not more
`than 3.8 mm, a diameter of the openings of the wells is not
`more than 4 mm and an inter-well spacing is usually industry 55
`standard, i.e. 4.5 mm. Usually the plates are vacuumformed
`in 36 well (6x6), 64 well (8x8) or 96 well (8x12) formats.
`As shown in FIG. 1, handling of the plate (1) containing
`multiple wells (2) is facilitated, by a rigid 0.5-1 mm thick
`plastic frame (3) which is heat bonded to the plate. However,
`for small format plates (36 and 64 well format) the plate
`including the frame is usually produced as one single piece
`during vacuum forming. The forming cycle is usually very
`short, i.e. 15-20 seconds. This allows even a manual pro­
`duction of approximately 1000 plates per person in 8 hours 65
`using one single mold vacuumforming device. The tempera­
`ture of small samples (3-10 µl) contained in ultrathin-walled
`
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`sure of 30--50 g per well without cracking the conically
`shaped walls. In contrary to the high pressure heated lid
`described in U.S. Pat. No. 5,508,197, the lid described here
`seals individual wells but not the edges of plate only.
`Therefore, even a single sample per multiwell plate can be
`amplified without sample loss. The tight thermal contact
`between the extremely thin walls of the wells and the body
`of the block ( 4) is achieved automatically by the increased
`air pressure arising in the sealed wells at elevated tempera­
`tures. The high pressure heated lid includes the screw
`mechanism (6), a heated metal plate (7) and a thermoinsu­
`lating gasket (8) isolating the sample block ( 4) from the
`metal plate (7). Conventionally, the metal plate (7) is heated
`by resistive heating, it's temperature is sensed by a ther­
`mistor (9) and controlled by the programmable controller
`(10). The gasket (8) is usually a 1.5-2 mm thick silicon­
`rubber gasket. It serves for a tight pressuring of a sealing
`film (11) to the top surface of the multiwell plate (1) and for
`the thermal isolation of the sample block ( 4 ) from the metal
`plate (7). The sealing film (11) is usually a 50 micron-thick
`polypropylene film. Surprisingly, by the above means of
`sealing the plates, samples of a volume of as few as, for
`example, 0.5 µl can be easily amplified without reducing the
`PCR efficiency.
`For comparison, conventional, low-pressure heated lid
`(U.S. Pat. No. 5,475,610) and high pressure heated lid (U.S.
`Pat. No. 5,508,197) can be reliably used for oil-free tem­
`perature cycling of samples of a minimum volume of 15
`µl-20 µ1. However, it is clear that the use of ultra thin-walled
`microplates with elastic walls according to industry­
`standard formats and the method of sealing as described in
`FIG. 2 also improves the performance of conventional heat
`block thermocyclers in size and speed. To obtain a sufficient
`rigidity the plates can be formed, for example, out of
`reinforced plastic films by means of, for example, matched­
`die forming (stamping,-shaped rubber tool forming, hydro­
`forming or other technologies. Furthermore, such plates can
`also be formed as two-piece parts, in which the frame (3)
`supports not only the edges of the plate but also individual
`wells (2). In this case, the height of the wells has to be
`measured from the bottom side of the frame. Such frames
`can be produced as skirted frames suitable for robotic
`applications.
`Rapid heat block temperature cycler according to the
`invention (FIG. 2) was experimentally tested for the ampli­
`fication of a 455-base pairs long fragment of human papil­
`loma virus DNA The sample volume was 3 µ1. The
`temperature/time profile used for temperature cycling is
`shown in FIG. 3. The samples (i.e. standard PCR-mixtures
`without any carrier molecules) were transferred into the
`wells of the plate by means of conventional pipetting
`equipment. The plate was covered by sealing film (11),
`transferred into the heatblock of the thermocycler and tightly
`sealed by the heated lid as shown in FIG. 2. Upon sealing,
`a number of 30 PCR cycles was performed in 10 minutes
`using the temperature/time profile shown in FIG. 3. The
`heating rate was 10° C. per second, the cooling rate was 6°
`C. per second. The PCR product was analyzed by conven­
`tional agarose electrophoresis. The 455-base pairs long
`DNA fragment was amplified with a high specificity at the
`indicated ramping rates (supra).
`Summarized, this invention has many advantages when
`compared to capillary or microfabricated rapid thermocy­
`clers. Multiple small-volume samples can be easily loaded
`into the wells of ultrathin-walled multiwell plate by con­
`ventional pipetting equipment. Furthermore, they can be
`rapidly and efficiently sealed by using a high-pressure
`
`10
`
`20
`
`15
`
`6
`
`heated lid. Upon amplification the samples can be easily
`recovered for product analysis by electrophoresis or
`hybridization, thus allowing also high throughput amplifi­
`cation. Finally, standard PCR mixtures can be used for rapid
`temperature cycling without adding carriers, like BSA Last
`but not least, the use of disposable, inexpensive, ultrathin­
`walled plates allows a great reduction of the total costs. It is
`obvious that the rapid heat block thermocycler according to
`the present invention can fabricated in various formats, i.e.
`multiblock thermocyclers, exchangable block
`thermocyclers, temperature gradient thermocyclers and oth­
`ers. Furthermore, it is obvious that it can be produced to
`perform the reactions in highsample density plates, such as
`384-well plates or others.
`The following example serves to illustrate the invention
`but should not be construed as a limitation thereof. Example:
`A heat block thermocycler for subjecting a plurality of
`samples to rapid thermal cycling according to the invention
`is depicted in FIG. 2, wherein
`1) is a 36-well plate
`2) is a 16 µl well
`3) is a 0.5-mm thick plastic frame
`4) is a 3 cmx3 cm sample block (with a thermal mass of 4,5
`Joules/K)
`25 5) is a 3-µl sample
`6) is a screw mechanism of the heated lid
`7) is a heated bronze plate (thickness: 5 mm)
`8) is a thermoinsulating, 1.5 mm thick silicon-rubber gasket
`9) is a termistor
`10) is a programmable controller
`11) is a 50 µm thick polypropylene sealing film
`12) is a 57-watt thermoelectric module (3 cmx3 cm; Peltier
`module)
`13) is an air cool copper heat sink (540 Joules/K)
`14) is a thermocouple with a response time of approximately
`0.01 second.
`
`What we claim:
`
`1. A heat block thermocycler for subjecting plurality of
`samples to rapid thermal cycling, the heat block thermocy­
`cler comprising:
`a means for holding the plurality of samples including:
`a deformable ultrathin-walled multiwell plate having
`an array of conically shaped wells with a wall
`thickness at a thickest part of the wells of not more
`than 50 µm; and
`a low profile, low thermal mass and low thermal
`capacity sample block having an array of similarly
`shaped wells, wherein a height of the wells of said
`deformable ultrathin-walled multiwell plate is not
`more than a height of said low profile, low thermal
`mass and low thermal capacity sample block;
`a means for heating and cooling said low profile, low
`thermal mass and low thermal capacity sample block
`including at least one thermoelectric module; and
`a means for sealing the plurality of samples including a
`high pressure, moveable, heated lid.
`2. A heat block thermocycler according to claim 1,
`wherein said deformable ultrathin-walled multiwell plate
`has a thinnest part in a bottom of each well.
`3. A heat block thermocycler according to claim 1,
`wherein said deformable ultrathin-walled multiwell plate
`has a thickness at a thinnest part in the range of 15 µm to 20
`µm.
`4. A heat block thermocycler according to claim 3,
`65 wherein said low profile, low thermal mass and low thermal
`capacity sample block has a thermal capacity of not more
`than 6 watt seconds per 0 C.
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`THERMO FISHER EX. 1041
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`US 6,556,940 Bl
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`5. A heat block thermocycler according to claim 1,
`wherein each well of said deformable ultrathin-walled mul­
`tiwell plate has a volume of not more than 40 µ1.
`6. A heat block thermocycler according to claim 1,
`wherein said low profile, low thermal mass and low thermal
`capacity sample block has a height of not more than 4 mm.
`7. A heat block thermocycler according to claim 6,
`wherein said low profile, low thermal mass and low thermal
`capacity sample block has a thermal capacity of not more
`than 6 watt seconds per 0 C.
`8. A heat block thermocycler according to claim 7,
`wherein said low profile, low thermal mass and low thermal
`capacity sample block has a thermal mass of 4.5 Joules/K.
`9. A heat block thermocycler according to claim 8,
`wherein said low profile, low thermal mass and low thermal 15
`capacity sample block is designed for biological samples of
`1 µl-20 µ1.
`10. A heat block thermocycler according to claim 1,
`wherein said low profile, low thermal mass and low thermal
`capacity sample block has a thermal capacity of not more 20
`than 6 watt seconds per 0 C.
`11. A heat block thermocycler according to claim 10,
`wherein said low profile, low thermal mass and low thermal
`capacity sample block has a thermal mass of 4.5 Joules/K.
`12. A heat block thermocycler according to claim 11, 25
`wherein said low profile, low thermal mass and low thermal
`capacity sample block is designed for biological samples of
`1 µl-20 µ1.
`13. A heat block thermocycler according to claim 1,
`wherein said low profile, low thermal mass and low thermal 30
`capacity sample block has a thermal mass of 4.5 Joules/K.
`14. A heat block thermocycler according to claim 13,
`wherein said low profile, low thermal mass and low thermal
`capacity sample block is designed for biological samples of
`1 µl-20 µ1.
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`8
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`15. A heat block thermocycler according to claim 1,
`wherein said low profile, low thermal mass and low thermal
`capacity sample block is designed for biological samples of
`1 µl-20 µ1.
`16. A heat block thermocycler according to claim 1,
`wherein temperature of said low profile, low thermal mass
`and low thermal capacity sample block is rapidly and
`controllably increased and decreased at a rate of at least as
`great as 5° C. per second by a single thermoelectric module.
`17. A heat block thermocycler according to claim 1,
`wherein force of the high pressure, moveable, heated lid is
`applied to said low profile, low thermal mass and low
`thermal capacity sample block.
`18. A heat block thermocycler according to claim 1
`wherein force of the high pressure, moveable, heated lid is
`applied to portions of said deformable ultrathin-walled mul­
`tiwell plate lying between said wells of said low profile, low
`thermal mass and low thermal capacity sample block to seal
`the wells.
`19. A heat block thermocycler according to claim 1,
`wherein force of the high pressure, moveable, heated lid is
`applied to portions of said deformable ultrathin-walled mul­
`tiwell plate lying between said wells of said low profile, low
`thermal mass and low thermal capacity sample block to seal
`the wells and is not more than 100 Kg per total surface.
`20. A heat block thermocycler according to claim 1,
`wherein the high pressure, moveable, heated lid includes an
`elastic insulating gasket.
`21. A heat block thermocycler according to claim 1,
`wherein the high pressure, moveable, heated lid includes a
`silicon rubber gasket.
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`THERMO FISHER EX. 1041
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