Patent EP0283593A1 successfully refreshed from Google Patents.
US5656493A
Expired / Lapsed
US
⏱️ 0.0 years remaining
System for automated performance of the polymerase chain reaction
Assignee: Applied Biosystems LLC
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Filing: 1994-02-18
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Granted: 1997-08-12
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Expires: 2014-02-18
Abstract
Abstract There is disclosed herein a machine for performing nucleic acid amplification under computer control. The machine utilizes any one of a number of heating and cooling systems under control of a host computer which directs the heating and cooling systems to heat and cool a reaction-chamber-containing heat exchanger at appropriate times in the process. The reaction chambers are pre-loaded with the nucleic acid(s) to be amplified, a thermostable enzyme to catalyze polymerization, specific oligonucleotide primers, and four different nucleotide triphosphates. Also disclosed is the process for the amplification chain reaction implemented by the machine, which utilizes a thermostable enzyme.
Inventors
- 👤 Kary B. Mullis
- 👤 Larry Johnson
- 👤 Richard A. Leath
- 👤 Timothy J. Wennberg
- 👤 Louis M. Mezei
- 👤 Joseph T. Widunas
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-06 16:00
CPC Classifications
B; B01; B01L; B01L7/00; B01L7/52; B01J; B01J19/00; B01J19/0046; C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6844; C12Q1/686; B01J2219/00; B01J2219/00274; B01J2219/00277; B01J2219/00495; B01J2219/00583; B01J2219/0059; B01J2219/0068; B01J2219/00686; B01J2219/00689; B01J2219/00718; B01J2219/0072; B01J2219/00722; C40; C40B; C40B40/00; C40B40/04; C40B40/06; C40B60/00; C40B60/14
Claims Analysis
Total Claims: 18
Claim 1
Independent Claim
168 words
A thermal cycling system for performing a polymerase chain reaction amplification protocol comprising multiple cycles of the steps of thermal denaturation of double-stranded DNA, primer hybridization to single-stranded DNA, and template-dependent primer extension by a DNA polymerase, comprising: at least one reaction mixture comprising at least one single- or double-stranded nucleic acid sequence to be amplified, four different deoxyribonucleotides, and a pair of oligodeoxyribonucleotide primers for each said at least one nucleic acid sequence to be amplified, for each said at least one reaction mixture, a heat-conducting reaction chamber, in thermal contact with each said at least one chamber, a variable temperature heating and cooling system, the temperature of said heating and cooling system being computer controllable, and a user-initiable computer controllingly coupled to said heating and cooling system, said computer being programmed to vary the temperature of said heating and cooling system and thereby to vary the temperature of said at least one chamber in accordance with said polymerase chain reaction protocol upon initiation by a user.
Claim 2
Dependent Claim
168 words
A thermal cycling system for performing a polymerase chain reaction amplification protocol comprising multiple cycles of the steps of thermal denaturation of double-stranded DNA, primer hybridization to single-stranded DNA, and template-dependent primer extension by a DNA polymerase, comprising: at least one reaction mixture comprising at least one single- or double-stranded nucleic acid sequence to be amplified, four different deoxyribonucleotides, and a pair of oligodeoxyribonucleotide primers for each said at least one nucleic acid sequence to be amplified, for each said at least one reaction mixture, a heat-conducting reaction chamber, in thermal contact with each said at least one chamber, a variable temperature heating and cooling system, the temperature of said heating and cooling system being computer controllable, and a user-initiable computer controllingly coupled to said heating and cooling system, said computer being programmed to vary the temperature of said heating and cooling system and thereby to vary the temperature of said at least one chamber in accordance with said polymerase chain reaction protocol upon initiation by a user.
Claim 3
Dependent Claim
38 words
The thermal cycling system of claim 1, wherein said multiple cycles of the polymerase chain reaction protocol include a repetitive cycle which includes denaturation in the range of from 90° to 105° C. for up to 4 minutes.
Claim 4
Dependent Claim
16 words
The thermal cycling system of claim 2, wherein said cycle is repeated at least 15 times.
Claim 5
Dependent Claim
69 words
The thermal cycling system of claim 1 comprising a plurality of reaction mixtures, each in a separate reaction chamber, and wherein said heating and cooling system includes a metal block having a plurality of recesses shaped to fit said chambers and fluid flow channels, a temperature-controlled cooling fluid reservoir, and controllable pumping apparatus for pumping a cooling fluid from said cooling fluid reservoir through said channels in said block.
Claim 6
Dependent Claim
93 words
The thermal cycling system of claim 4, wherein said multiple cycles of the polymerase chain reaction protocol include a repetitive cycle which includes bring said chamber to a hybridization temperature in the range of from 35° to 65° C. for from 0.5 to 5 minutes, followed by extension product synthesis at a temperature in the range of from 40° to 80° C. for from 0.5 to 40 minutes and then by heating said chamber to a denaturation temperature in the range of from 90° to 105° C. for from 0.5 to 4 minutes.
Claim 7
Dependent Claim
16 words
The thermal cycling system of claim 5, wherein said cycle is repeated at least 15 times.
Claim 8
Dependent Claim
63 words
The thermal cycling system of claim 4, wherein said multiple cycles of the polymerase chain reaction protocol include a repetitive cycle which includes bringing said chamber to a hybridization temperature in the range of from 35° to 65° C., followed by heating said chamber to a denaturation temperature in the range of from 90° to 105° C. for from 0.5 to 4 minutes.
Claim 9
Dependent Claim
16 words
The thermal cycling system of claim 7, wherein said cycle is repeated at least 15 times.
Claim 10
Dependent Claim
39 words
The thermal cycling system of claim 4, wherein said heating and cooling system comprises a temperature-controlled heating fluid reservoir and controllable pumping apparatus for pumping a heating fluid from said heating fluid reservoir through said channels in said block.
Claim 11
Dependent Claim
41 words
The thermal cycling system of claim 4, further comprising a computer-controlled liquid handler having at least one reagent container and pipettes for transferring reagent from said at least one reagent container into said chamber in response to a transfer control signal.
Claim 12
Dependent Claim
17 words
The thermal cycling system of claim 4, wherein said heating and cooling system comprises in electrical heater.
Claim 13
Dependent Claim
35 words
The thermal cycling system of claim 1, wherein said heating and cooling system has the capability to cool said at least one reaction mixture to temperatures suitable for reactions utilizing E. coli DNA polymerase I.
Claim 14
Dependent Claim
38 words
The thermal cycling system of claim 1, wherein said heating and cooling system has the capability to cool said at least one reaction mixture to temperatures suitable for reactions utilizing Klenow fragment of E. coli DNA polymerase I.
Claim 15
Dependent Claim
20 words
The thermal cycling system of claim 1, wherein said steps of hybridization and extension are performed at the same temperature.
Claim 16
Dependent Claim
23 words
The thermal cycling system of claim 1, wherein said step of extension is performed at a temperature higher than said step of hybridization.
Claim 17
Dependent Claim
44 words
The thermal cycling system of claim 1 comprising a plurality of reaction mixtures and a plurality of reaction chambers, and wherein said heating and cooling system includes a metal block having a plurality of recesses shaped to fit said chambers and a Peltier device.
Claim 18
Dependent Claim
16 words
The thermal cycling system of claim 1, wherein said programmed computer is user-programmable in real time.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2000-08-11 | 2001-02-10 | 2001-08-12 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2004-08-11 | 2005-02-10 | 2005-08-12 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2008-08-11 | 2009-02-10 | 2009-08-12 | $7700.00 | Window Closed / Lapsed |