United States Patent [19]
`Atwood et al.
`
`llllllllllllllllllllllllllllllllllllllllllIllllllllllllllllllllllllllllllll
`USOO547561OA
`5,475,610
`Patent Number:
`Dec. 12, 1995
`Date of Patent:
`
`[11]
`[45]
`
`[54]
`
`[75]
`
`THERMAL CYCLER FOR AUTOMATIC
`PERFORMANCE OF THE POLYlVIERASE
`CHAIN REACTION WITH CLOSE
`TEMPERATURE CONTROL
`
`Inventors: John G. Atwood, West Redding;
`Albert C. Mossa, Trumbull; Lisa M.
`Goven, Bridgeport; Fenton Williams,
`Brook?eld; Timothy M. Woudenberg,
`Bethel, all of Conn.; Marcel
`Margoulies, Scarsdale, N.Y.; Robert P.
`Ragusa, Newton, Conn.; Richard
`Leath, Berkley; Clive Miles, San
`Rafael, both of Calif.
`
`[73]
`
`Assignee: The Perkin-Elmer Corporation,
`Norwalk, Conn.
`
`[21]
`[22]
`
`Appl. No.:
`
`Filed:
`
`871,264
`Apr. 20, 1992
`
`[63]
`
`[51]
`[52]
`
`[53]
`
`[56]
`
`Related U.S. Application Data
`
`Continuation-impart of Ser. No. 620,606, Nov. 29, 1990,
`abandoned, and Ser. No. 670,545, Mar. 14, 1991, aban
`doned.
`
`Int. Cl.6 ..................................................... .. G06G 7/58
`U.S. Cl. ........................ .. 364/500; 364/496; 364/497;
`364/498; 364/499; 165/12
`Field of Search ................................... .. 364/166, 183,
`364/496-500, 557; 236/46; 435/52, 290;
`422/88; 361/380; 65/1, 12
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`Re. 34,133 11/1992 Thorne .................................... .. 422/99
`3,311,303
`3/1967 Noyes.
`3,392,914 7/1968 Nienstadt.
`
`(List continued on next page.)
`
`FOREIGN PATENT DOCUMENTS
`
`0134622 5/1983 European Pat. Off. .
`171140 5/1984 European Pat. OE. .
`
`0128778 12/1984 European Pat. Off. .
`0223618 7/1985 European Pat. Off. .
`
`(List continued on next page.)
`
`OTHER PUBLICATIONS
`
`Techne TP-16 Instruction Manual (16 pages), 1990.
`Techne TP-16 Advertisement (2 pages), Dec. 1984.
`Techne TP-l6 Operating Instruction Manual, May 1984.
`Coy Laboratory Products Inc., Model 50/60, Tempcycler
`Manual, dated Jul. 11, 1991.
`Histomat advertisement, R. Jung GmbH, Oct., 1980.
`Lake Shore Cryotronics, Inc., Review of Scienti?c Instru
`ments, Jul. 1980.
`Barber-Colman Co. UP55 Setpoint Programmer, 1980.
`Techne Brochure, on PHC—2 1982.
`
`(List continued on next page.)
`
`Primary Examiner—-Emanuel T. Voeltz
`Assistant Examiner-Kamini Shah
`Atzomey, Agent, or Firm—Davis Hoxie Faithfull & Hap
`good
`
`ABSTRACT
`[57]
`An instrument for performing highly accurate PCR employ
`ing a sample block in rnicrotiter tray format. The sample
`block has local balance and local symmetry. A three zone
`?lm heater controlled by a computer and ramp cooling
`solenoid valves also controlled by the computer for gating
`coolant ?ow through the block controls the block tempera
`ture. Constant bias cooling is used for small changes.
`Sample temperature is calculated instead of measured. A
`platen deforms plastic caps to apply a minimum acceptable
`threshold force for seating the tubes and thermally isolates
`them. A cover isolates the block. The control software
`includes diagnostics. An install program tests and charac
`terizes the instrument. A new user interface is used. Dispos
`able, multipiece plastic microtiter trays to give individual
`freedom to sample tubes are taught.
`
`167 Claims, 39 Drawing Sheets
`
`Micro?che Appendix Included
`(10 Micro?che, 890 Pages)
`
`I HEATED COVER '“14
`SAMPLES
`I10
`
`22
`LT\
`
`21»
`
`T‘MPERAT
`16_ USER'S
`L
`uRE
`PROGRAMMED -I 2
`KEYBOARD
`SAMPLE BLOCK
`AND DISPLAY
`I\_
`20
`l56—~ FILM HEATER
`~—/28
`__
`\26
`HEATED COVER
`18
`x
`30_,l l 32_,l l
`I CONTROL
`coNTRoL
`_52
`SAMPLE BLOCK
`COMPUTER AND
`gggrLRAgL"
`POWER SUPPLIES I HEATER coNTRoLs
`63
`R91 E16 :COOLANT CONTROLS |-=' SYSTEM
`L
`54)
`’ 50
`5.
`
`r58)
`
`7’
`
`: u
`i 5
`, g
`I E
`
`74
`
`~36
`l
`
`34
`I 46 NFREON [48
`38
`4o
`\ REFRIGERATION
`r
`UNlT
`::
`
`42
`
`44
`
`THERMO FISHER EX. 1042
`
`

`

`5,475,610
`Page 2
`
`US. PATENT DOCUMENTS
`
`OTHER PUBLICATIONS
`
`3,483,997 12/1969 Ritter ...................................... .. 211/76
`
`P, S, Martin, et 31,, J_ Parent Sci Tech, date unknown,
`
`
`
`Ct 3,856,471 12/1974 VVmitz.
`
`......................... ..
`
`R
`
`
`
`Peltier heat pumps from Matcrials Electronics unknown_
`
`3,912,913 10/1975 Bummg -
`3,983,363
`9/1976 Alter .
`4,008,048 2/1977 Hellemans et al. .
`4,206,872
`6/1980 Levine.
`4,312,835
`1/1982 201m et a1_ _
`4,335,620
`6/1982 Adams.
`4,362,699 12/1982 Verlander.
`4,401,594
`8/1983 Umezawa et a1. ................. .. 260/112.5
`4,404,345
`9/1933 schl'enkel' -
`4,474,015 10/1984 Christmas et a1. .
`4’478’094 10,1984 Saloma et a1‘ '
`4,483,823 10/1984 Umetsu .
`4,504,733
`3/1985 Walsh.
`4,517,160
`5,1985 Gane et a1_ _
`4,518,700
`5/1985 Stephens _
`4,534,941
`811985 Stephens 61111..
`4,544,436 11/1985 Chlosta et al. .
`4,554,839 11/1985 Hewlett et a1. .
`4,593,049 7/1986 Zelinka -
`
`Techne Ad for on Block PHC—1, date unknown.
`t
`T hn TP__16 T
`t
`Pr
`Ad rt.
`6° 6
`ampera “re
`°grammer
`"6 ‘Semen
`d?“ unknowP-
`_
`B1ores b.v.B1oexcellence TAQ-Polymerase and Ampliclone
`Kit date unknown
`Techne PHC—1 Ad date unknown.
`Techne PHC—2 Temperature Cycler Ad date unknown.
`Techne Flow Coolers FC-200 and FC200 and Dip Cooler
`RU_2O0 date unknown
`Techne Tempunit ro Tempette Immersion Circulators Ad
`date unknown
`.
`'
`Dlalog Search for Techne Patents date unknown:
`Forma Sc1ent1?c Advert1sement, Analytlc Chennstry, Aug.
`8. 1982. Brook?eld Test Chamber.
`Cole-Partner Instr. Co. 1985-1986 Catalog.
`IEEE Transactions on Biomedical Engineering, vol.
`BME—29, No. 8, Aug. 1982, pp. 555-568 V. J. Anselmo et
`al. “Programmable Temperature Control System for Bio
`
`’
`
`’
`
`.
`
`.
`
`ijflll‘lljfteflal
`1123:1132‘
`4,683,202 7/1987 Mullis.
`4,685,081
`8/1937 Richman _
`4,703,836 11/19g7 Nelson_
`4,711,851 12/1987 McNamara et a1. .
`4,800,159
`l/1989 Mullis et al. ...................... .. 435/1723
`4,858,155
`8/1989 Okawa et a1. .
`4,865,986
`9/1989 Coy et a1. ............................. .. 436/290
`4,889,818 12/1989 Gelfand et a1. ....................... .. 435/194
`léayq et a1‘ '
`4,965,188 10,1990 Mums et a1_ _
`4,981,801
`1/1991 Suzuki et a1_ _
`5,008,182 4/1991 Sninsky et al. ........................... .. 435/5
`5,038,852
`8/1991 Johnson et a1. . . . . . . . . . .
`. . . .. 165/12
`5,056,427 10/1991 Sakabe ....... ..
`100/211
`5,075,216 12/1991 Innis e181-
`----- -- 435/6
`5,167,929 12/1992 Korf et al. ............................ .. 422/102
`
`,
`
`,
`
`shimoto ............................. .. 435/290
`
`FOREIGN PATENT DOCUMENTS
`
`0164054
`0200362
`0236069
`0238313
`0325763
`03114-40
`0318255
`0388159
`2413708
`2359422
`2490632
`2490362
`2650593
`2603683
`88 08 738.7
`57-098013
`60-24188
`62-12986
`664094
`2161815
`8807297
`8909437
`89/12502
`90/08298
`910636
`
`12/1985
`12/1986
`9/1987
`9/1987
`2/1989
`4/1989
`5/1989
`9/1990
`12/1977
`2/1978
`3/1982
`3/1982
`5/1977
`8/1977
`10/1988
`6/1982
`4/1985
`6/1989
`2/1988
`1/1986
`3/1988
`10/1989
`12/1989
`7/1990
`5/1991
`
`European Pat. Off. .
`European Pat. Off. .
`European Pat. Off. .
`European Pat. O?. .
`European Pat. Off. .
`European Pat. Off. .
`European Pat. O?“. .
`European Pat. Off. .
`France .
`
`France .
`
`France .
`
`France .
`
`Germany .
`Germany .
`Germany .
`Japan .
`Japan .
`Japan .
`
`Switzerland .
`United Kingdom .
`
`WIPO .
`
`WIPO .
`
`WIPO .
`
`WIPO .
`
`WIPO .
`
`logical Materials”.
`
`.
`
`,
`
`,,
`
`Amino Acid Analysis System Rev. Sci. Instrum. 51(7), Jul.
`‘1,980;
`_
`_
`_
`Stud1es on Polynucleot1des-The Linkage of Deoxyn
`bopolynucleotide Templates to Cellulose and Its Use in
`Their Replication” by Panet and Khorara, The Journal of
`Biological Chemistry, V01. 249, N0. 16, Issue ofAug. 25, pp.
`5213_5221 (1974)_
`“ Advances in Laboratory Automation Robotics 1984” by
`Zynark Corp
`“Studies on Polynucleotides—Repair Replication of Short
`Synthetic DNA s As Catalyzed by DNA Polymerase by
`Kleppe 6t 31. .1. M01. B101. (1971) 56, pp. 341-461.
`Studies on Polymlcleotidesqotal Synthesis of the Structural
`Gene For An Alanic Transfer Ribonucleic Acid from Yeast,
`by Khorana et al. J. Mol. Biol. (1972) 72, PP. 209-217.
`Studies on Polynucleotides-Hybridization of Polydeoxy
`nucleotides with Tryosine Transfer RNA Sequences to the 1'
`Strand 080 psu DNA, by Miller et al., J. Mol. Biol, (1972)
`72, pp. 593-522.
`“Automation of Microliter Plate Chromogenic Substrate
`LAL Endotoxin Assay Method by use of a Modi?ed Pro/
`Pette Express System”, Martin et al. J. Parent Sci Tech, vol.
`40, No. 2, pp. 61-66, Man-Apr. 1986.
`Saiki et al., “Enzymatic Ampli?cation of B-Globin
`Genomic Sequences and Restriction Site Analysis for Diag
`nosis of Sickle Cell Anemia”, Science vol. 320 pp.
`1350-1354, Dec. 1985.
`Tecam Dry Heat Baths, Catalog 7051081 date unknown.
`Techne (OB-1) Block Digestor) Catalog 7051091 date
`unknown.
`Techne (Dri-Block 08-3), Catalog 7051101 date unknown.
`Brook?eld Test Chamber, advertisement, Date unknown.
`Gene Machines Open Markets, S. Russell, San Francisco
`Chromical Jan. 11, 1988.
`PTC—100 Programmable Thermal Controller, MJ Research,
`Inc. date unknown.
`Techne PHC—1 Dri-Block Operating Instructions date
`unknown.
`Thermoelectric Coolers Tackle Jobs Heat Jobs Heat Sinks
`Can’t—.l. McDermott, EDN May 20, 1980.
`Laboratory Methods “A Programmable System to Perform
`
`THERMO FISHER EX. 1042
`
`

`

`5,475,610
`Page 3
`
`the Polymerase Chain Reaction”, Weier, et al. date
`unknown.
`Nucleic Acids Research, vol. 16, No. 7, 1988; Rollo et a1.
`Nucleic Acids Research, vol. 16, No. 12, 1988; Foulkes, et
`al.
`Frigichip—Miniature Ceramic Modules, Series FC, Melcor
`date unknown.
`Model 2010 and 2011 Controllers Pu, P, LFE Corporation
`date unknown.
`PC Application Ideas, Instruments & Control, Oct. 1980,
`Jack Hickey.
`Bicoent Model Name: ROB 3, ROB 4, Advertisement date
`unkown.
`Biodesign Systems, Model Name: Therrno 4, Advertisement
`date unkown.
`Bio Med, Model Name: PCR Processor; Thermocycler 10;
`Thermocycler 60, Advertisements and description of
`PCR—Processor by Oers.
`BioMelIa, Model Name: Trio notes of Aug. 7, 1989.
`Biotherrn Corp., Developer Biotherm, Model Name:
`
`BioOven, advertisement Jun. 1989.
`B. Braun, Model Name: Thermocycler, Advertisement date
`unknown.
`Chromatec, Inc., Model Name: TAC 3000a, notes to Jon
`Raymond from Fenton Williams of Perkin Elmer re New
`Cycler Competitor and Letter of Nov. 29, 1989 from Richard
`Messerschmidt of Chromotec, Inc. to Jona Laboratories.
`Coy Labs, Model Name: TempCycler, Distributor: Mepco
`Scienti?c, Rotech Scienti?c Inst., advertisement, J. Fenton
`William May 12, 1989 “Competitive Analysis: Coy Labo
`ratories Model 50 Tempcyler”, Perkin Elmer memo to BIO
`Competitor Hotline Distribution from B. Delisle of Aug. 3,
`1989 re Coy TempCycler.
`Dale Designs, Developer; Dale Designs, Model Name:
`Genesmaid, Distributor; Dale Designs description of
`“Genesmaid: cycling water bath”.
`Dalton, Model Name: Programmable Cnzyme Reactor,
`questionnaire date unknown.
`Eppendorf, Inc., Model Name: MicroCycler, advertisement
`Jan. 1, 1989.
`
`THERMO FISHER EX. 1042
`
`

`

`US. Patent
`
`Dec. 12, 1995
`
`O_.mun—IEE<mEE>ou82%.H
`
`53mmas/waAI
`fizz/Egan._mmiémmzfi
`
`NN
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`
`
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`
`mm>OUembed:
`
`155.200
`
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`
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`
`A,I!.m5[Vn:2:
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`Qm<0m>m¥
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`405.200
`
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`
`F.OE
`
`THERMO FISHER EX. 1042
`
`THERMO FISHER EX. 1042
`
`
`
`
`
`
`
`

`

`US. Patent
`
`Dec. 12, 1995
`
`Sheet 2 of 39
`
`5,475,610
`
`
`
`THERMO FISHER EX. 1042
`
`THERMO FISHER EX. 1042
`
`

`

`US. Patent
`
`Dec. 12, 1995
`
`Sheet 3 of 39
`
`5,475,610
`
`
`
`
`
`
` 19MJJMMM.
`
`
`107
`116
`100
`
`FIG.6~
`
`
`
`Y
`
`X
`
`THERMO FISHER EX. 1042
`
`THERMO FISHER EX. 1042
`
`

`

`US. Patent
`
`Dec. 12, 1995
`
`Sheet 4 0f 39
`
`5,475,610
`
`Q12S
`
`o
`
`[12
`Q124
`
`116
`FIG. 4.1
`
`X
`
`Y’
`
`THERMO FISHER EX. 1042
`
`

`

`US. Patent
`
`Dec. 12, 1995
`
`Sheet 5 0f 39
`
`5,475,610
`
`[78
`
`(121
`
`u
`
`[78
`
`<2;
`
`K116
`
`FIG. 7
`
`(1’20
`
`u
`‘Hg
`
`Y
`
`I
`
`66
`
`/78
`
`[12
`
`67
`
`U
`
`Q; @399 (P Q)
`
`K98 107/ k116 L99
`971-,‘ 4166} 70-)
`FIG. 8
`rIY
`
`0w
`
`2
`
`12217
`( 78 218
`
`219 {220
`
`224
`221 (223 225
`
`12
`
`78
`
`146
`226
`
`I
`
`0
`
`:
`
`o
`
`l
`
`o
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`0
`
`I
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`6
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`°
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`l 10x09?’ I
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`°
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`‘Q
`
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`u 160 g 150
`
`1 54
`
`1 52
`
`J
`
`FIG. 9
`
`1483 2
`
`Y
`
`X
`
`THERMO FISHER EX. 1042
`
`

`

`US. Patent
`
`Dec.12,1995
`
`Sheet 6 of 39
`
`5,475,610
`
`VOLTS
`
`TEMP.
`100- C‘.
`
`so'c;
`
`so'C-
`
`25 C"
`
`+
`
`164
`166
`
`V
`
`<—-—->
`
`P
`
`TIME
`
`162
`
`FIG. 1 O
`
`DENATURATION
`
`EXTENSION
`
`"
`
`‘
`
`‘I
`
`174
`
`HYBRIDIZATION
`
`FIG. 1 1
`
`p
`TIME
`
`THERMO FISHER EX. 1042
`
`

`

`U.S. Patent
`
`Dec. 12, 1995
`
`Sheet 7 of 39
`
`5,475,610
`
`PLASTIC COOLANT
`MANIFOLD
`Z60
`/
`MANIFOLD HEATER
`- l- _ _ _ _ _ _ _ _ - "' - T -b156
`O:
`
`W'- 266
`
`gl
`ml
`w
`g
`
`CENTRAL HEATER
`I
`
`254
`
`I5
`I
`luJ
`L’)
`‘a
`
`L L _ _ __ _ _ _ _ _ _ _ _ l \
`
`/ MANIFOLD HEATER
`256
`PLASTIC COOLANT
`MANIFOLD
`
`\ \_ 258
`\ 262
`\
`\' 268
`
`FIG. 13
`
`SAMPLE
`TEMP.
`
`T
`
`TIME
`
`THERMO FISHER EX. 1042
`
`

`

`US. Patent
`
`Dec. 12, 1995
`
`Sheet 8 of 39
`
`5,475,610
`
`SAMPLE
`TEMP.
`
`"-51
`
`‘ 286
`
`TIME
`
`FIG. 14.1
`
`THERMO FISHER EX. 1042
`
`

`

`US. Patent
`
`Dec. 12, 1995
`
`Sheet 9 0f 39
`
`5,475,610
`
`/‘~..
`( 346
`W4
`‘m 2::
`a
`1 W376
`J g ,
`12
`“352
`f 384 280
`288
`f
`/ 27s—\ 278)
`.
`
`l
`
`Q 29
`
`l
`156 // / // / / / / /§*~115
`
`0/~1’\ \ \ \ \ \ \ \ \ \
`16
`
`FIG. 15
`
`THERMO FISHER EX. 1042
`
`

`

`US. Patent
`
`Dec. 12, 1995
`
`Sheet 10 of 39
`
`5,475,610
`
`VOLTS
`
`VOLTS
`
`294
`
`V1
`
`TIME
`
`TIME
`
`FIG. 16A
`
`FIG. 1 65
`
`WEIGHTING
`
`FIG. 16D
`
`1:1 T2T3'I'4T5
`FIG. 16C
`
`TIME
`
`THERMO FISHER EX. 1042
`
`

`

`US. Patent
`
`Dec. 12, 1995
`
`Sheet 11 of 39
`
`5,475,610
`
`SAMPLE TEMP - 6 WELLS
`
`94.6
`94.4
`94.2
`94
`93.8
`93.6
`93.4
`93.2
`93
`
`92.8
`92.6
`92.4
`92.2
`92
`
`80
`
`90
`
`FIG. 17
`
`100
`
`110
`
`AMOUNT OF DNA
`GENERATED
`
`FIG. 18
`
`THERMO FISHER EX. 1042
`
`

`

`US. Patent
`
`Dec. 12, 1995
`
`Sheet 12 of 39
`
`5,475,610
`
`319
`320
`
`\ \ \
`
`314 ~
`
`147
`
`324
`
`332 V
`
`336
`
`320
`
`THERMO FISHER EX. 1042
`
`

`

`US. Patent
`
`Dec. 12, 1995
`
`Sheet 13 of 39
`
`5,475,610
`
`564
`
`Q
`
`\
`\
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`
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`
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`
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`
`576
`
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`
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`
`THERMO FISHER EX. 1042
`
`

`

`US. Patent
`
`5,475,610
`
`I
`949*»?QWWMmMmmMmfi
`
`
`©++++++
`
`.-
`
`FIG. 22
`
`THERMO FISHER EX. 1042
`
`THERMO FISHER EX. 1042
`
`

`

`_
`
`US. Patent
`
`5,475,610
`
`
`00 00
`OO
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`FIG. 23
`
`THERMO FISHER EX. 1042
`
`

`

`U.S. Patent
`
`Dec. 12,1995
`
`Sheet 16 of 39
`
`5,475,610
`
`/346
`
`\\ 342
`
`FIG. 24
`
`p 346
`
`1
`
`/
`
`/346
`
`372
`
`FIG. 25
`
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`
`THERMO FISHER EX. 1042
`
`

`

`US. Patent
`
`Dec. 12, 1995
`
`Sheet 17 of 39
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`5,475,610
`
`346»;
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`
`

`

`US. Patent
`
`Dec. 12, 1995
`
`Sheet 18 of 39
`
`5,475,610
`
`
`
`THERMO FISHER EX. 1042
`
`THERMO FISHER EX. 1042
`
`

`

`US. Patent
`
`Dec. 12, 1995
`
`Sheet 19 of 39
`
`5,475,610
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`

`

`US. Patent
`
`Dec. 12, 1995
`
`Sheet 21 of 39
`
`5,475,610
`
`.410
`
`FIG. 36
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`THERMO FISHER EX. 1042
`
`THERMO FISHER EX. 1042
`
`

`

`US. Patent
`
`Dec. 12, 1995
`
`Sheet 22 of 39
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`5,475,610
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`

`US. Patent
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`5,475,610
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`FIG. 39
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`THERMO FISHER EX. 1042
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`
`

`

`US. Patent
`
`Dec. 12, 1995
`
`Sheet 24 of 39
`
`5,475,610
`
`FIG. 41
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`FIG. 40
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`THERMO FISHER EX. 1042
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`THERMO FISHER EX. 1042
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`

`

`US. Patent
`
`Dec. 12,1995
`
`Sheet 25 of 39
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`

`US. Patent
`
`Dec. 12, 1995
`
`Sheet 26 of 39
`
`5,475,610
`
`FIG. 44
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`
`
`FIG. 46
`
`THERMO FISHER EX. 1042
`
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`
`
`

`

`US. Patent
`
`Dec. 12, 1995
`
`Sheet 27 of 39
`
`5,475,610
`
`
`
`FIG. 45 '
`
`THERMO FISHER EX. 1042
`
`THERMO FISHER EX. 1042
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`

`

`US. Patent
`
`Dec. 12, 1995
`
`Sheet 28 of 39
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`5,475,610
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`

`US. Patent
`
`Dec. 12, 1995
`
`Sheet 30 of 39
`
`5,475,610
`
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`

`US. Patent
`
`Dec. 12, 1995
`
`Sheet 31 of 39
`
`5,475,610
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`

`

`US. Patent
`
`Dec. 12, 1995
`
`Sheet 32 of 39
`
`5,475,610
`
`594
`
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`
`THERMO FISHER EX. 1042
`
`

`

`US. Patent
`
`Dec. 12, 1995
`
`Sheet 33 of 39
`
`5,475,610
`
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`

`US. Patent
`
`Dec. 12, 1995
`
`Sheet 34 of 39
`
`5,475,610
`
`1 DO
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`
`FIG. 51
`
`THERMO FISHER EX. 1042
`
`THERMO FISHER EX. 1042
`
`

`

`US. Patent
`
`Dec. 12, 1995
`
`Sheet 35 of 39
`
`5,475,610
`
`RESET
`
`KEY TO
`FIG. 53
`
`CLEAR
`REMOTE
`FLAG
`
`FIG. 53 (1)
`
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`
`THERMO FISHER EX. 1042
`
`THERMO FISHER EX. 1042
`
`

`

`US. Patent
`
`Dec. 12, 1995
`
`Sheet 36 of 39
`
`5,475,610
`
`INSTR NOT CALIBRATE
`
`CALL SERVICE
`LOCKUP KEYPAD
`
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`
`YES
`
`FIG. 53 (2)
`
`THERMO FISHER EX. 1042
`
`THERMO FISHER EX. 1042
`
`

`

`US. Patent
`
`Dec. 12, 1995
`
`Sheet 37 of 39
`
`5,475,610
`
`0
`
`NO
`
`INSTR. NOT
`INSTALLED
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`
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`
`FIG. 54
`
`THERMO FISHER EX. 1042
`
`THERMO FISHER EX. 1042
`
`

`

`US. Patent
`
`Dec. 12, 1995
`
`Sheet 38 of 39
`
`5,475,610
`
`F/G55./ -
`
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`
`
`
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`
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`
`
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`
`THERMO FISHER EX. 1042
`
`THERMO FISHER EX. 1042
`
`

`

`US. Patent
`
`Dec. 12, 1995
`
`Sheet 39 of 39
`
`5,475,610
`
`(10 Microfiche, 890 Pages)
`
`INSTR NOI
`
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`THERMO FISHER EX. 1042
`
`THERMO FISHER EX. 1042
`
`

`

`1
`THERMAL CYCLER FOR AUTOMATIC
`PERFORMANCE OF THE POLYMERASE
`CHAIN REACTION WITH CLOSE
`TEMPERATURE CONTROL
`
`This is a continuation-in-part application of U.S. patent
`application Ser. No. 07/620,606, filed Nov. 29, 1990 by
`Mossa et al. and U.S. patent application Ser. No. 07/670,545,
`filed Mar. 14, 1991, both of which are hereby incorporated
`herein by reference both abandoned. Microfiche Appendi-
`cies C—F are attached, including 10 sheets of microfiche
`comprising 890 frames.
`The invention pertains to the field of computer directed
`instruments for performing the polymerase chain reaction
`(hereafter PCR). More particularly, the invention pertains to
`automated instruments that can perform the polymerase
`chain reaction simultaneously on many samples with a very
`high degree of precision as to results obtained for each
`sample. This high precision provides the capability, among
`other things, of performing so-called “quantitative PCR”.
`To amplify DNA (Deoxyribose Nucleic Acid) using the
`PCR process, it is necessary to cycle a specially constituted
`liquid reaction mixture through a PCR protocol including
`several different temperature incubation periods. The reac—
`tion mixture is comprised of various components such as the
`DNA to be amplified and at least two primers selected in a
`predetermined way so as to be sufficiently complementary to
`the sample DNA as to be able to create extension products
`of the DNA to be amplified. The reaction mixture includes
`various enzymes and/or other reagents, as well as several
`deoxyribonucleoside triphosphates such as dATP, dCTP,
`dGTP and dTTP. Generally, the primers are oligonucleotides
`which are capable of acting as a point of initiation of
`synthesis when placed under conditions in which synthesis
`of a primer extension product which is complimentary to a
`nucleic acid strand is induced,
`i.e.,
`in the presence of
`nucleotides and inducing agents such as thermostable DNA
`polymerase at a suitable temperature and pH.
`The Polymerase Chain Reaction (PCR) has proven a
`phenomenally successful technology for genetic analysis,
`largely because it is so simple and requires relatively low
`cost
`instrumentation. A key to PCR is the concept of
`thermocycling: alternating steps of melting DNA, annealing
`short primers to the resulting single strands, and extending
`those primers to make new copies of double stranded DNA.
`In thermocycling, the PCR reaction mixture is repeatedly
`cycled from high temperatures (>90° C.) for melting the
`DNA, to lower temperatures (40° C. to 70° C.) for primer
`annealing and extension. The first commercial system for
`performing the thermal cycling required in the polymerase
`chain reaction,
`the Perkin—Elmer Cetus DNA Thermal
`Cycler, was introduced in 1987.
`Applications of PCR technology are now moving from
`basic research to applications in which large numbers of
`similar amplifications are routinely run. These areas include
`diagnostic
`research,
`biopharmaceutical
`development,
`genetic analysis, and environmental testing. Users in these
`areas would benefit from a high performance PCR system
`that would provide the user with high throughput, rapid
`tum-around time, and reproducible results. Users in these
`areas must be assured of reproducibility from sample-to-
`sample,
`run—to-run,
`lab-to-lab, and instrument-to—instru-
`ment.
`
`For example, the physical mapping process in the Human
`Genome Project may become greatly simplified by utilizing
`sequence tagged sites. An STS is a short, unique sequence
`easily amplified by PCR and which identifies a location on
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`5,475,610
`
`2
`
`the chromosome. Checking for such sites to make genome
`maps requires amplifying large numbers of samples in a
`short time with protocols which can be reproducibly run
`throughout the world.
`As the number of PCR samples increases, it becomes
`more important
`to integrate amplification with sample
`preparation and post—amplification analysis. The sample
`vessels must not only allow rapid thermal cycling but also
`permit more automated handling for operations such as
`solvent extractions and centrifugation. The vessels should
`work consistently at low volumes, to reduce reagent costs.
`Generally PCR temperature cycling involves at least two
`incubations at different temperatures. One of these incuba-
`tions is for primer hybridization and a catalyzed primer
`extension reaction. The other incubation is for denaturation,
`i.e., separation of the double stranded extension products
`into single strand templates for use in the next hybridization
`and extension incubation interval. The details of the poly-
`merase chain reaction, the temperature cycling and reaction
`conditions necessary for PCR as well as the various reagents
`and enzymes necessary to perform the reaction are described
`in U.S. Pat. Nos. 4,683,202, 4,683,195, EPO Publication
`258,017 and U.S. Pat. No. 4,889,818 (Taq polymerase
`enzyme patent), which are hereby incorporated by reference.
`The purpose of a polymerase chain reaction is to manu-
`facture a large volume of DNA which is identical to an
`initially supplied small volume of “seed” DNA. The reaction
`involves copying the strands of the DNA and then using the
`copies to generate other copies in subsequent cycles. Under
`ideal conditions, each cycle will double the amount of DNA
`present thereby resulting in a geometric progression in the
`volume of copies of the “target” or “seed” DNA strands
`present in the reaction mixture.
`A typical PCR temperature cycle requires that the reac-
`tion mixture be held accurately at each incubation tempera—
`ture for a prescribed time and that the identical cycle or a
`similar cycle be repeated many times. A typical PCR pro-
`gram starts at a sample temperature of 94° C. held for 30
`seconds to denature the reaction mixture. Then, the tem-
`perature of the reaction mixture is lowered to 37° C. and held
`for one minute to permit primer hybridization. Next, the
`temperature of the reaction mixture is raised to a tempera-
`ture in the range from 50° C. to 72° C. where it is held for
`two minutes to promote the synthesis of extension products.
`This completes one cycle. The next PCR cycle then starts by
`raising the temperature of the reaction mixture to 94° C.
`again for strand separation of the extension products formed
`in the previous cycle (denaturation). Typically, the cycle is
`repeated 25 to 30 times.
`Generally, it is desirable to change the sample tempera-
`ture to the next temperature in the cycle as rapidly as
`possible for several reasons. First, the chemical reaction has
`an optimum temperature for each of its stages. Thus, less
`time spent at nonoptimum temperatures means a better
`chemical result is achieved. Another reason is that a mini-
`
`mum time for holding the reaction mixture at each incuba-
`tion temperature is required after each said incubation
`temperature is reached. These minimum incubation times
`establish the “floor” or minimum time it takes to complete
`a cycle. Any time transitioning between sample incubation
`temperatures is time which is added to this minimum cycle
`time. Since the number of cycles is fairly large, this addi-
`tional time unnecessarily lengthens the total time needed to
`complete the amplification.
`In some prior automated PCR instruments, the reaction
`mixture was stored in a disposable plastic tube which is
`closed with a cap. A typical sample volume for such tubes
`
`THERMO FISHER EX. 1042
`
`THERMO FISHER EX. 1042
`
`

`

`3
`
`4
`
`5,475,610
`
`was approximately 100 microliters. Typically, such instru-
`ments used many such tubes filled with sample DNA and
`reaction mixture inserted into holes called sample wells in a
`metal block. To perform the PCR process, the temperature of
`the metal block was controlled according to prescribed
`temperatures and times specified by the user in a PCR
`protocol file. A computer and associated electronics then
`controlled the temperature of the metal block in accordance
`with the user supplied data in the PCR protocol file defining
`the times, temperatures and number of cycles, etc. As the
`metal block changed temperature, the samples in the various
`tubes followed with similar changes in temperature. How-
`ever, in these prior art instruments not all samples experi-
`enced exactly the same temperature cycle. In these prior art
`PCR instruments, errors in sample temperature were gener-
`ated by nonuniformity of temperature from place to place
`within the metal sample block, i.e., temperature gradients
`existed within the metal of the block thereby causing some
`samples to have diiferent temperatures than other samples at
`particular times in the cycle. Further, there were delays in
`transferring heat from the sample block to the sample, but
`the delays were not the same for all samples. To perform the
`PCR process successfully and efliciently, and to enable so
`called “quantitative” PCR, these time delays and tempera-
`ture errors must be minimized to a great extent.
`The problems of minimizing time delays for heat transfer
`to and from the sample liquid and minimizing temperature
`errors due to temperature gradients or nonuniformity in
`temperature at various points on the metal block become
`particularly acute when the size of the region containing
`samples becomes large. It is a highly desirable attribute for
`a PCR instrument to have a metal block which is large
`enough to accommodate 96 sample tubes arranged in the
`format of an industry standard microtiter plate.
`The microtiter plate is a widely used means for handling,
`processing and analyzing large numbers of small samples in
`the biochemistry and biotechnology fields. Typically, a
`microtiter plate is a tray which is 3% inches wide and 5
`inches long and contains 96 identical sample wells in an 8
`well by 12 well rectangular array on 9 millimeter centers.
`Although microtiter plates are available in a wide variety of
`materials, shapes and volumes of the sample wells, which
`are optimized for many different uses, all microtiter plates
`have the same overall outside dimensions and the same 8x12
`array of wells on 9 millimeter centers. A wide variety of '
`equipment is available for automating the handling, process—
`ing and analyzing of samples in this standard microtiter plate
`format.
`Generally microtiter plates are made of injection molded
`or vacuum formed plastic and are inexpensive and consid-
`ered disposable. Disposability is a highly desirable charac-
`teristic because of the legal liability arising out of cross
`contamination and the difficulty of washing and drying
`microtiter plates after use.
`It is therefore a highly desirable characteristic for a PCR
`instrument to be able to perform the PCR reaction on up to
`96 samples simultaneously said samples being arranged in a
`microtiter plate format.
`Of course, the size of the metal block which is necessary
`to heat and cool 96 samples in an 8X12 well array on 9
`millimeter centers is fairly large. This large area block
`creates multiple challenging engineering problems for the
`design of a PCR instrument which is capable of heating and
`cooling such a block very rapidly in a temperature range
`generally from 0° to 100° C. with v