(12) United States Patent
`Brown et al.
`
`USOO673O883B2
`(10) Patent No.:
`US 6,730,883 B2
`(45) Date of Patent:
`May 4, 2004
`
`(54) FLEXIBLE HEATING COVER ASSEMBLY
`FOR THERMAL CYCLING OF SAMPLES OF
`BIOLOGICAL MATERIAL
`
`(75) Inventors: Larry R. Brown, Carlsbad, CA (US);
`William D. Brumley, Vista, CA (US);
`Kenneth J. Zajac, San Diego, CA (US)
`(73) Assignee: Stratagene, La Jolla, CA (US)
`
`(*) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days
`a --
`y U dayS.
`
`(21) Appl. No.: 10/262,994
`(22) Filed:
`Oct. 2, 2002
`(65)
`Prior Publication Data
`US 2004/0065655 A1 Apr. 8, 2004
`
`5,656,493 A 8/1997 Mullis et al. ............ 435/286.1
`5,710,381 A
`1/1998 Atwood et al. .......... 73/864.91
`5,779,981 A
`7/1998 Danssaert et al. ............ 422/99
`5,785,926 A 7/1998 Seubert et al. .............. 422/100
`List continued
`t
`(List continued on next page.)
`FOREIGN PATENT DOCUMENTS
`438883
`7/1991
`............ B01D/1/30
`488769
`6/1992 ............ C12O/1/68
`O955097 A1 * 11/1999
`O5-168459
`7/1993 ............ C12M/1/OO
`O7-3O8183
`11/1995
`..... ... C12M/1/OO
`09/322755
`12/1997 ..... ... C12M/1/OO
`WO 98/12502
`12/1989
`............. BO1 L/7/OO
`WO 98/43740
`10/1998 ............. BO1 L/7/OO
`WO OO/32312
`6/2000 ............. BO1 L/7/OO
`
`EP
`EP
`EP
`JP
`JP
`JP
`WO
`WO
`WO
`
`OTHER PUBLICATIONS
`KR
`“Multi-functional
`temperature
`circulator,
`2001038297A, abstract and single figure, May 15, 2001.*
`
`(51) Int. CI.7 - - - - - - - - - - - - - - - - - - - - - - - - - - - C12M 1/00; C12M 1/38;
`
`Primary Examiner Joseph Pelham
`
`(56)
`
`U.S. PATENT DOCUMENTS
`
`(74) Attorney, Agent, or Firm-Palmer & Dodge, LLP,
`H05B3/20; H05B 3/34; G05D 23/20
`Kathleen M. Williams; David J. Dykeman
`(52) U.S. Cl. ....................... 219/428; 219/385; 219/521;
`219/530; 219/535; 219/549; 435/286.1;
`(57)
`ABSTRACT
`435/288.4; 435/304.4
`(58) Field of Search ......................... 5 - - - - - - 5. E. A flexible heating cover assembly for an apparatus for
`54 Eo's s s 1. 2.5 3, 4.
`heating Samples of biological material with Substantial tem
`s
`/289.1,
`• Is
`• us
`•-1s
`perature uniformity includes a housing having a plurality of
`References Cited
`engageable enclosure components, a resistive heater having
`a plurality of heater element areas, a heater backing plate
`providing Stability to the resistive heater, a force distribution
`System that distributes a force over the heater backing plate;
`and a Support plate providing stiffness for the force distri
`3. A 3.E. R. O. - - - - - - - - - - "...
`bution System, wherein the arrangement of the resistive
`4,865,986 A 9/1989 Coy et al......
`... 435/290
`heater, the heater backing plate, the force distribution System
`4.950,608 A 8/1990 Kishimoto ....
`... 435/290
`and the Support plate provide Substantial temperature uni
`5,038,852 A 8/1991 Johnson et al. ............... 165/12
`formity among a plurality of Sample tubes for receiving
`5,061,630 A 10/1991 Knopf et al. .....
`... 435/290
`Samples of biological material. The flexible heating cover
`5.255,976 A 10/1993 Connelly ..................... 374/31
`assembly improves the uniformity, efficiency, quality, reli
`5,333,675 A 8/1994 Mullis et al. ................. 165/12
`ability and controllability of the thermal response during
`5.3% A 2. E. SC al - - - - - -
`3.
`5552,580 A g:
`yet at Of: thermal cycling of the biological material.
`5,602,756 A 2/1997 Atwood et al. ......
`... 364/500
`5,616,301 A 4/1997 Moser et al. ............... 422/104
`
`
`
`82 Claims, 28 Drawing Sheets
`
`Agilent Exhibit 1218
`Page 1 of 50
`
`

`

`US 6,730,883 B2
`Page 2
`
`U.S. PATENT DOCUMENTS
`
`9/1998 Okuda et al. ................. 62/3.7
`5,813,233 A
`5,819,842 A 10/1998 Potter et al. ..
`... 165/206
`s
`A . 3. R al.
`45.
`2Y-- a-2
`allile . . . . . . . . . . . . . . . . . . .
`5,928.907 A
`7/1999 Woudenberg et al. ..... 435/91.2
`6,004,512 A 12/1999 Titcomb et al. .....
`... 422/63
`6,054,263 A 4/2000 Danssaert et al. ............. 435/4
`
`
`
`6,093,370 A 7/2000 Yasuda et al. ............. 422/68.1
`6,106,784. A 8/2000 Lund et al. ....
`... 422/104
`6,153,426 A * 11/2000 Heimberg ...
`435/305.4
`6,337,435 B1
`1/2002 Chu et al. ................... 136/242
`6,638,761 B2 * 10/2003 Shin et al. ............... 435/288.4
`
`6,657,169 B2 12/2003 Brown ....................... 219/385
`
`* cited by examiner
`
`Agilent Exhibit 1218
`Page 2 of 50
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`

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`U.S. Patent
`
`May 4, 2004
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`Sheet 1 of 28
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`US 6,730,883 B2
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`Page 3 of 50
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`U.S. Patent
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`May 4, 2004
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`US 6,730,883 B2
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`U.S. Patent
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`May 4, 2004
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`FIG. 3
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`Agilent Exhibit 1218
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`U.S. Patent
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`May 4, 2004
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`:
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`U.S. Patent
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`May 4, 2004
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`Sheet 6 of 28
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`US 6,730,883 B2
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`
`
`
`Go
`G) (G) G)
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`O
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`O
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`Agilent Exhibit 1218
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`U.S. Patent
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`May 4, 2004
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`Sheet 7 of 28
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`US 6,730,883 B2
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`4-NY
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`Agilent Exhibit 1218
`Page 9 of 50
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`U.S. Patent
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`May 4, 2004
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`US 6,730,883 B2
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`US. Patent
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`May 4, 2004
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`Sheet 10 of 28
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`US 6,730,883 B2
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`S.
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`Agilent Exhibit 1218
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`May 4, 2004
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`May 4, 2004
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`U.S. Patent
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`May 4, 2004
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`Sheet 18 of 28
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`US 6,730,883 B2
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`
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`Agilent Exhibit 1218
`Page 20 of 50
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`U.S. Patent
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`May 4, 2004
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`Sheet 19 of 28
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`US 6,730,883 B2
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`O O O O O O O O O O O O O O O O O O O O O O O O O O O C) O O O O O O O O ?-O O O OO O O O O O O O O O O OVO O O O O O O O O O O O|O O O O O O O O
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`Agilent Exhibit 1218
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`U.S. Patent
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`May 4, 2004
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`Sheet 20 0f 28
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`US 6,730,883 B2
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`360
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`FIG. 27
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`May 4, 2004
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`Sheet 21 of 28
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`US 6,730,883 B2
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`360
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`Agilent Exhibit 1218
`Page 23 of 50
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`U.S. Patent
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`May 4, 2004
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`Sheet 22 of 28
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`US 6,730,883 B2
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`-350
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`00000 /
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`Agilent Exhibit 1218
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`U.S. Patent
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`May 4, 2004
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`Sheet 23 of 28
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`US 6,730,883 B2
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`350 300
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`
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`O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O C) O O O O O O
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`
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`Agilent Exhibit 1218
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`U.S. Patent
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`May 4, 2004
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`Sheet 24 of 28
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`US 6,730,883 B2
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`408
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`U.S. Patent
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`May 4, 2004
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`Sheet 28 of 28
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`US 6,730,883 B2
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`550
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`F.G. 40
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`550
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`FIG. 41
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`Agilent Exhibit 1218
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`US 6,730,883 B2
`
`1
`FLEXBLE HEATING COVER ASSEMBLY
`FOR THERMAL CYCLING OF SAMPLES OF
`BIOLOGICAL MATERIAL
`
`RELATED APPLICATIONS
`
`None.
`
`FIELD OF THE INVENTION
`The present invention relates to a heating cover assembly
`for an apparatus for heating Samples of biological material,
`and more particularly to a flexible heating cover assembly
`that improves the uniformity, efficiency, quality, reliability
`and controllability of the thermal response during thermal
`cycling of DNA samples to accomplish a polymerase chain
`reaction, a quantitative polymerase chain reaction, a reverse
`transcription-polymerase chain reaction, or other nucleic
`acid amplification types of experiments.
`
`15
`
`BACKGROUND OF THE INVENTION
`Techniques for thermal cycling of DNA samples are
`known in the art. By performing a polymerase chain reaction
`(PCR), DNA can be amplified. It is desirable to cycle a
`Specially constituted liquid biological reaction mixture
`through a specific duration and range of temperatures in
`order to successfully amplify the DNA in the liquid reaction
`mixture. Thermal cycling is the process of melting DNA,
`annealing Short primers to the resulting Single Strands, and
`extending those primers to make new copies of double
`stranded DNA. The liquid reaction mixture is repeatedly put
`through this process of melting at high temperatures and
`annealing and extending at lower temperatures.
`In a typical thermal cycling apparatus, a biological reac
`tion mixture including DNA will be provided in a large
`number of sample wells on a thermal block assembly. It is
`desirable that the samples of DNA have temperatures
`throughout the thermal cycling process that are as uniform
`as reasonably possible. Even Small variations in the tem
`perature between one sample well and another Sample well
`can cause a failure or undesirable outcome of the experi
`ment. For instance, in quantitative PCR, one objective is to
`perform PCR amplification as precisely as possible by
`increasing the amount of DNA that generally doubles on
`every cycle; otherwise there can be an undesirable degree of
`disparity between the amount of resultant mixtures in the
`Sample wells. If Sufficiently uniform temperatures are not
`obtained by the Sample wells, the desired doubling at each
`cycle may not occur. Although the theoretical doubling of
`DNA rarely occurs in practice, it is desired that the ampli
`fication occurs as efficiently as possible.
`In addition, temperature errors can cause the reactions to
`improperly occur. For example, if the Samples are not
`controlled to have the proper annealing temperatures, certain
`forms of DNA may not extend properly. This can result in
`the primers in the mixture annealing to the wrong DNA or
`not annealing at all. Moreover, by ensuring that all Samples
`are uniformly heated, the dwell times at any temperature can
`be shortened, thereby speeding up the total PCR cycle time.
`By shortening this dwell time at certain temperatures, the
`lifetime and amplification efficiency of the enzyme are
`increased. Therefore, undesirable temperature errors and
`variations between the Sample well temperatures should be
`decreased.
`Prior art heating covers used in PCR heating equipment
`are simple, Stiff, and relatively inexpensive. The prior art
`
`25
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`2
`designs have mainly involved a Stiff metal plate, a simple
`resistive heater, and an insulating cover. Because quantita
`tive data was not generated, the heating covers did not have
`to control condensation in the biological Samples as pre
`cisely as the heating covers used in QPCR equipment. Also,
`because optical data was not collected, the prior art heating
`cover designs were not complicated with the need to provide
`a means to excite and collect the optical data through the
`heating cover. Prior art heating covers used in QPCR heating
`equipment are mainly derived from their earlier PCR coun
`terparts that provide a means for optical Signal transmission,
`but, prior art heating covers are Still mainly Stiff designs
`which do not provide a uniform force distribution about the
`Sample containers.
`Prior art heating covers are difficult to use, expensive,
`complicated and do not provide uniform thermal contact or
`uniform force distribution about the sample wells. U.S. Pat.
`No. 5,475,610 discloses an instrument for performing PCR
`employing a cover which can be raised or lowered over a
`sample block. U.S. Pat. No. 5,475,610 does not disclose a
`cover assembly that is flexible to provide a more uniform
`thermal contact and force distribution on the Sample tube
`caps. U.S. Pat. No. 5,928,907 discloses a system for carrying
`out real time fluorescence-based measurements of nucleic
`acid amplification products. U.S. Pat. No. 5,928,907 does
`not disclose a cover assembly that is flexible to provide a
`more uniform thermal contact and force distribution on the
`Sample tube caps. The prior art does not disclose a cover
`assembly that is flexible to provide a more uniform thermal
`contact and force distribution on the Sample tube caps.
`In light of the foregoing, there is a need in the art for a
`flexible heating cover assembly that enhances the thermal
`response uniformity, efficiency, quality, reliability and con
`trollability of the DNA sample wells in the thermal cycling
`apparatuS.
`
`SUMMARY OF THE INVENTION
`The present invention is a flexible heating cover assembly
`that improves the uniformity, efficiency, quality, reliability
`and controllability of the thermal response during thermal
`cycling of DNA samples to accomplish a polymerase chain
`reaction, a quantitative polymerase chain reaction, a reverse
`transcription-polymerase chain reaction, or other nucleic
`acid amplification types of experiments.
`The present invention is a flexible heating cover assembly
`for an apparatus for heating Samples of biological material
`with Substantial temperature uniformity including a housing
`having a plurality of engageable enclosure components, a
`resistive heater located within the housing, the resistive
`heater including a plurality of heater element areas, a heater
`backing plate engaging the resistive heater and providing
`protection and Stability to the resistive heater, a force
`distribution System that engages the heater backing plate and
`distributes a force over the heater backing plate; and a
`Support plate providing Stiffness for the force distribution
`System, wherein the arrangement of the resistive heater, the
`heater backing plate, the force distribution System and the
`Support plate provide Substantial temperature uniformity
`among a plurality of Sample tubes for receiving Samples of
`biological material. The flexible heating cover assembly
`improves the uniformity, efficiency, quality, reliability and
`controllability of the thermal response during thermal
`cycling of DNA samples.
`In another aspect of the present invention, the resistive
`heater produces a non-uniform heat distribution along a
`Surface exposed to the plurality of Sample tubes. The resis
`
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`Page 31 of 50
`
`

`

`3
`tive heater further comprises a plurality of heater element
`areas including at least one outer heater element area and at
`least one central heater element area.
`In another aspect of the present invention, the heater
`backing plate is thin to promote flexibility when the heater
`backing plate is connected to the resistive heater. The heater
`backing plate is composed of a thermally conductive mate
`rial.
`In another aspect of the present invention, the force
`distribution System further comprises at least one Spring
`Strip and a Spring retainer plate. The at least one Spring Strip
`has an elongated body and a plurality of Spring extensions to
`distribute the force uniformly on the heater backing plate.
`In another aspect of the present invention, the Support
`plate has Sufficient Stiffness to provide a reaction force for
`the force distribution system with minimal deflection of the
`Support plate.
`In another aspect of the present invention, the resistive
`heater, the heater backing plate, and the Support plate each
`comprise a plurality of aligned Sample well openings, each
`Sample well opening corresponding to a respective Sample
`tube of the plurality of sample tubes.
`The present invention is a flexible heating cover assembly
`with enhanced functions including the flexibility of the
`cover assembly and the force distribution. In addition, the
`flexible heating cover assembly of the present invention
`enables the resistive heater to float in a vertical direction, So
`that the resistive heater has Some freedom of movement
`Vertically which leads to a more uniform thermal contact and
`force distribution and more accurate and consistent results.
`The flexible heating cover assembly of the present invention
`provides thermal insulation for the upper portion of the
`Sample tubes and the Sample caps.
`BRIEF DESCRIPTION OF THE DRAWINGS
`The accompanying drawings, which are incorporated in
`and constitute a part of this specification, illustrate Several
`embodiments of the invention and together with the
`description, Serve to explain the principles of the invention.
`The present invention will be further explained with refer
`ence to the attached drawings, wherein like Structures are
`referred to by like numerals throughout the several views.
`The drawings shown are not necessarily to Scale, with
`emphasis instead generally being placed upon illustrating
`the principles of the present invention.
`FIG. 1 is a top perspective view of a flexible heating cover
`assembly of the present invention.
`FIG. 2 is a bottom perspective view of a flexible heating
`cover assembly of the present invention.
`FIG. 3 is a perspective view of a flexible heating cover
`assembly of the present invention attached to an apparatus
`for thermally cycling Samples of a biological material.
`FIG. 4 is a front sectional view of a flexible heating cover
`assembly of the present invention attached to an apparatus
`for thermally cycling Samples of a biological material.
`FIG. 5 is a partial enlarged front sectional view of a
`flexible heating cover assembly of the present invention.
`FIG. 6 is a top view of a thermal block assembly of a
`thermal System base.
`FIG. 7 is a perspective view of a thermal block assembly
`of a thermal System base.
`FIG. 8 is a perspective sectional view of a sample well of
`a thermal System base.
`FIG. 9 is a perspective view of a sensor cup of a thermal
`System base.
`
`15
`
`25
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
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`US 6,730,883 B2
`
`4
`FIG. 10 is a perspective view of a heat sink of a thermal
`System base.
`FIG. 11 is a bottom view of a heat sink of a thermal
`System base.
`FIG. 12 is a top view of a solid state heater a heat sink of
`a thermal System base.
`FIG. 13 is a side view of a Solid state heater a heat sink
`of a thermal System base.
`FIG. 14 is a perspective view of a solid state heater of a
`thermal System base.
`FIG. 15 is a top view of a spacer bracket with a solid state
`heater of a thermal System base.
`FIG. 16 is a top perspective view of a spacer bracket of
`a thermal System base.
`FIG. 17 is a bottom perspective view of a spacer bracket
`of a thermal System base.
`FIG. 18 is a top view of a heat sink, a bottom resistive
`heater, and a plurality of Solid State heaters of a thermal
`System base.
`FIG. 19 is a bottom view of a thermal block plate and a
`plurality of Solid State heaters of a thermal System base.
`FIG. 20 is a top exploded assembly view of a flexible
`heating cover assembly of the present invention showing
`how a Stiff Support plate, a Spring Strip, a Spring retainer
`plate, a heater backing plate, a plurality of heater Slides, a
`resistive heater, a cover assembly skirt interact with a
`plurality of biological Sample tubes having Sample caps.
`FIG. 21 is a bottom exploded assembly view of a flexible
`heating cover assembly of the present invention showing
`how a Stiff Support plate, a Spring Strip, a Spring retainer
`plate, a heater backing plate, a plurality of heater Slides, a
`resistive heater, a cover assembly skirt interact with a
`plurality of biological Sample tubes having Sample caps.
`FIG. 22 is a perspective view of a resistive heater of a
`flexible heating cover assembly of the present invention
`showing a layout of a plurality of heater element areas.
`FIG. 23 is a top perspective view of a resistive heater of
`a flexible heating cover assembly of the present invention
`showing a thermistor.
`FIG. 24 is a bottom perspective view of a resistive heater
`of a flexible heating cover assembly of the present invention
`showing a plurality of insulating pads.
`FIG. 25 is a top view of a resistive heater of a flexible
`heating cover assembly of the present invention showing a
`thermistor.
`FIG. 26 is a side view of a resistive heater of a flexible
`heating cover assembly of the present invention.
`FIG. 27 is a perspective view of a heater backing plate of
`a flexible heating cover assembly of the present invention.
`FIG. 28 is a top view of a heater backing plate of a flexible
`heating cover assembly of the present invention.
`FIG. 29 is a top perspective view of a resistive heater
`engaging a heater backing plate of a flexible heating cover
`assembly of the present invention.
`FIG. 30 is a bottom perspective view of a resistive heater
`engaging a heater backing plate of a flexible heating cover
`assembly of the present invention.
`FIG. 31 is a bottom view of a resistive heater engaging a
`heater backing plate of a flexible heating cover assembly of
`the present invention.
`FIG. 32 is a Side view of a resistive heater engaging a
`heater backing plate of a flexible heating cover assembly of
`the present invention.
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`Agilent Exhibit 1218
`Page 32 of 50
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`FIG.33 is a perspective view of a spring strip of a flexible
`heating cover assembly of the present invention.
`FIG. 34 is a top view of a spring strip of a flexible heating
`cover assembly of the present invention.
`FIG.35 is a side view of a spring strip of a flexible heating
`cover assembly of the present invention.
`FIG. 36 is a perspective View of a Spring retainer plate of
`a flexible heating cover assembly of the present invention.
`FIG.37 is a top view of a spring retainer plate of a flexible
`heating cover assembly of the present invention.
`FIG. 38 is a top perspective view of a stiff support plate
`of a flexible heating cover assembly of the present invention.
`FIG. 39 is a bottom perspective view of a stiff support
`plate of a flexible heating cover assembly of the present
`invention.
`FIG. 40 is a perspective view of a heater slide of a flexible
`heating cover assembly of the present invention.
`FIG. 41 is a front view of a heater slide of a flexible
`heating cover assembly of the present invention showing the
`U-shape of the preferred heater slide.
`While the above-identified drawings set forth preferred
`embodiments of the present invention, other embodiments
`of the present invention are also contemplated, as noted in
`the discussion. This disclosure presents illustrative embodi
`ments of the present invention by way of representation and
`not limitation. Numerous other modifications and embodi
`ments can be devised by those skilled in the art which fall
`within the Scope and Spirit of the principles of the present
`invention.
`
`15
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`25
`
`DETAILED DESCRIPTION
`A flexible heating cover assembly of the present invention
`is illustrated generally at 200 in FIGS. 1 and 2. As best
`shown in FIGS. 20 and 21, the flexible heating cover
`assembly 200 includes a cover assembly skirt 250, a resis
`tive heater 300, a heater backing plate 350, a spring strip
`400, a spring retainer plate 450, a stiff Support plate 500, and
`a plurality of heater slides 550. The flexible heating cover
`assembly 200 engages a plurality of biological Sample tubes
`140 having sample caps 146.
`As shown in FIG. 3, the flexible heating cover assembly
`200 can be attached to an apparatus for thermally cycling
`Samples of a biological material. The flexible heating cover
`assembly 200 can be attached to any apparatus for thermal
`cycling of DNA samples to accomplish a polymerase chain
`reaction, a quantitative polymerase chain reaction, a reverse
`transcription-polymerase chain reaction, or other nucleic
`acid amplification types of experiments. For example, the
`flexible heating cover assembly 200 can be attached to the
`apparatus for thermally cycling Samples of a biological
`material disclosed in assignee's co-pending U.S. patent
`application Ser. No. 09/364,051, the entirety of which is
`hereby incorporated by reference. When combined with a
`thermal system base 15 (which contains a thermal block
`assembly 20 for accepting Samples and means to heat and
`cool the thermal block assembly 20), the flexible heating
`cover assembly 200 improves the quality of the thermal
`response of the system for quantitative PCR.
`The thermal system base 15 includes a plurality of sample
`Wells for receiving Sample tubes of a biological reaction
`mixture. As shown in FIGS. 3-5, the thermal system base 15
`includes a thermal block assembly 20. Thermal block assem
`bly 20 includes a flat thermal block plate 22 and a plurality
`of sample wells 24 for receiving tubes with samples of
`DNA, as best shown in FIGS. 4, 6 and 7. Thermal block
`
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`US 6,730,883 B2
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`6
`plate 22 is Substantially rectangular and is of Sufficient size
`to accommodate a plurality of Sample wells 24 on the top
`Surface, but could be of other shapes (i.e., circular, oval,
`Square). In the embodiment shown in the drawings, the plate
`22 accommodates 96 Sample wells 24 in a grid having eight
`columns and twelve rows. The sample wells 24 are in an 8
`by 12 grid with center-to-center Spacing between adjacent
`sample wells 24 of about nine millimeters. In other embodi
`ments of the present invention, there may be more or leSS
`than 96 Sample wells, the Sample well arrangement may
`vary, and the center-to-center measurement between adja
`cent Sample wells 24 may be more or less than nine
`millimeters. It is to be understood that the number of sample
`Wells can be varied depending on the Specific application
`requirements. For example, the Sample wells could be
`arranged to form a grid which is sixteen by twenty-four,
`thereby accommodating 384 sample wells. The sample wells
`24 are conical in shape, as shown in FIG.8. The walls 25 of
`the tube are conical, and extend at an angle to the flat plate
`22. The bottom 26 of the interior of the sample well is
`rounded. The bottom of each sample well 24 is attached to
`the thermal block plate 22. It should be understood that the
`Sample wells 24 could have any shape (i.e., cylindrical,
`Square or similar shapes), So that the inner Surface of the
`sample wells 24 closely mates with the sample tube 140
`inserted inside.
`The Sample wells 24 are designed So that Sample tubes
`140 with DNA samples can be placed in the sample wells 24.
`FIG. 5 shows a partial cut-away croSS Section with Sample
`tubes 140 placed in the sample wells 24. Each sample well
`24 is sized to fit the sample tube 140 exterior so that there
`will be substantial contact area between the sample tube 140
`and the interior portion of a sample well wall 25 to enhance
`the heat transfer to the DNA sample in the sample tube 140
`and reduce differences between the DNA mixture and
`sample well temperatures. The sample tube 140 includes a
`conical wall portion 142 which closely mates with the
`sample well wall 25.
`The sample tubes 140 are available in three common
`forms: (1) Single tubes; (2) Strips of eight tubes which are
`attached to one another; and (3) tube trays with 96 attached
`Sample tubes. The present invention is preferably designed
`to be compatible with any of these three designs. The Sample
`tubes 140 may be composed of a plastic, preferably molded
`polypropylene, however, other Suitable materials are accept
`able. A typical sample tube 140 has a fluid volume capacity
`of approximately 200 ul, however other sizes and configu
`rations can be envisaged within the Spirit and Scope of the
`present invention. The fluid volume typically used in an
`experiment is substantially less than the 200 ul sample tube
`capacity.
`Although the preferred embodiment uses Sample wells,
`other Sample holding Structures Such as Slides, partitions,
`beads, channels, reaction chambers, Vessels, Surfaces, or any
`other Suitable device for holding a Sample can be envisaged.
`Moreover, although the preferred embodiment uses the
`Sample holding Structure for biological reaction mixtures,
`the Samples to be placed in the Sample holding Structure are
`not limited to biological reaction mixtures. Samples could
`include any type of product for which it is desired to heat
`and/or cool, Such as cells, tissues, microorganisms or non
`biological product.
`Alternatively, a thin film of clear or opaque material could
`be attached (to form a Seal) to the tops of the sample
`containers in place of a Series of caps. This type of Sample
`container cover can reduce the labor associated with cap
`installation for Some users. The flexible heating cover
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`Agilent Exhibit 1218
`Page 33 of 50
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`7
`assembly of the present invention works with this type of
`Sealed film container cover. Typically, these films are com
`posed of a thin plastic with a layer of epoxy which can be
`cured using heat, pressure, heat and pressure, or UV light.
`As embodied herein and shown for example in FIG. 5,
`each Sample tube 140 also has a corresponding Sample tube
`cap 146 for maintaining the biological reaction mixture in
`the Sample tube. The caps 146 are typically inserted inside
`a top cylindrical surface 144 of the sample tube 140. The
`caps 146 are relatively clear So that light can be transmitted
`through the cap 146. The sample tube caps 146 may be
`composed of a plastic, preferably molded polypropylene,
`however, other Suitable materials are acceptable. Each cap
`146 has an optical window 148 on the top surface of the cap.
`The optical window 148 in the cap 146 is thin, flat, com
`15
`posed of plastic, and allows radiation Such as excitation light
`to be transmitted to the DNA samples and emitted fluores
`cent light from the DNA to be transmitted back to an optical
`detection System during cycling.
`A biological probe can be placed in the DNA Samples So
`that fluorescent light is transmitted in and emitted out as the
`Strands replicate during each cycle. A Suitable optical detec
`tion System can detect the emission of radiation from the
`Sample. The detection System can thus measure the amount
`of DNA which has been produced as a function of the
`emitted fluorescent light. Data can be provided from each
`well and analyzed by a computer.
`As best shown in FIGS. 6 and 7, the thermal block plate
`22 is provided with mounting holes 27. Attachment screws
`or other fasteners pass through each of the mounting holes
`27. The arrangement of these fasteners will be discussed in
`greater detail below.
`AS best shown in FIGS. 6, 7, and 9, the thermal block
`assembly 20 further includes a plurality of sensor cups 28.
`The Sensor cups 28 are positioned adjacent the outer periph
`ery of the thermal block plate 22. In the illustrated
`embodiment, four Sensor cups 28 are positioned outside the
`grid of Sample wells 24. There is at least one Sensor cup for
`each thermoelectric or Solid State heating device used to heat
`the thermal block assembly 20. The details of the solid state
`heating devices will be discussed below. In the illustrated
`embodiment, four Solid State heating devices are used, and
`it is therefore appropriate to use at least fourthermal Sensors
`in the Sensor cups 28. If more Solid State heating devices
`were used, then it would be desirable to have more sensor
`cups 28. Each of the Solid State heating devices may heat at
`Slightly different temperatures, therefore the provision of a
`thermal Sensor in a Sensor cup 28 for each Solid State heater
`increases thermal block temperature uniformity.
`The sensor cups 28 each include a thermistor or other
`Suitable temperature Sensor positioned to measure the tem
`perature of the thermal