Device for thermo-dependent chain reaction amplification of target nucleic acid sequences, measured in real-time

• US 7,732,136 B2
• Filed: 08/25/2004
• Issued: 06/08/2010
• Est. Priority Date: 07/28/2000
• Status: Expired due to Fees

• Current Assignee: PALL Genesystems
• Original Assignee: PALL Genesystems
• Inventors: Festoc, Gabriel
• Primary Examiner(s): Forman; B J
• Assistant Examiner(s): MUMMERT, STEPHANIE KANE
• Application Number:
  US10/926,482
• Publication Number:
  US 20050026277A1
• Time in Patent Office: 2,113 Days
• Field of Search: None
• US Class Current: 435/6.14
• CPC Class Codes: B01L 2300/0803 Disc shape
• CPC Class Codes: B01L 2300/0809 rectangular shaped
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/1805 Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
• CPC Class Codes: B01L 2400/0406 capillary forces
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/049 vacuum
• CPC Class Codes: B01L 3/5025 for parallel transport of multiple samples
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/50273 characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: B01L 7/5255 by moving sample containers
• CPC Class Codes: B01L 7/54 using spatial temperature gradients

Abstract

The present invention concerns a device for amplifying target nucleic acids, reaction cartridges for use in the device, and modes of use of the device.

Claims

1. A method for amplifying a nucleic acid using a device comprising at least one circular cartridge having a plurality of reaction chambers and a reservoir, said reaction chambers being connected to the reservoir via channels, at least one heating plate having at least two distinct zones that can be heated to at least two different temperatures, wherein said distinct zones are sections of a disk or sections of a ring, and a means for relative rotational displacement between said at least one circular cartridge and said at least one heating plate, allowing a cyclic variation of the temperature of the reaction chambers wherein said means for relative rotational displacement is connected to said at least one circular cartridge for displacing the cartridge in relative rotation, said method comprising:

a) at least partially filling said reservoir with a fluid containing a sample of nucleic acids to be analyzed and reagents for carrying out an amplification reaction, with the exception of primers,

b) distributing said fluid to the reaction chambers of said at least one circular cartridge, in which are located the primers,

c) employing said means for relative rotational displacement to rotate said at least one circular cartridge to successively bring the contents of each reaction chamber to the at least two temperatures defined by the at least two distinct zones of said at least one heating plate,

wherein said nucleic acid is amplified.

2. The method according to claim 1, wherein said fluid further contains a fluorescent nucleic acid reporter.

3. The method according to claim 1, wherein said reaction chambers further comprise one or more probes.

4. The method according to claim 3, wherein said probes are labelled.

5. The method of claim 3, comprising further exciting and measuring the fluorescence of the contents of the reaction chambers in each cycle, with optical means for fluorescence excitation/measurement.

6. The method of claim 1, wherein c) comprises rotating said at least one circular cartridge while retaining in place said at least one heating plate.

7. The method of claim 1, wherein said at least one heating plate is constituted by two or three distinct independent heating zones.

8. The method of claim 1, wherein distributing fluid to the reaction chambers is carried out by applying an underpressure inside said at least one circular cartridge, then re-establishing the pressure.

9. A method for amplifying a nucleic acid using a device comprising at least one circular cartridge having a plurality of reaction chambers and a reservoir, said reaction chambers being connected to the reservoir via channels, at least one heating plate having at least two distinct zones that can be heated to at least two different temperatures, wherein said distinct zones are sections of a disk or sections of a ring, and a means for relative rotational displacement between said at least one circular cartridge and said at least one heating plate, allowing a cyclic variation of the temperature of the reaction chambers wherein said means for relative rotational displacement is connected to said at least one circular cartridge for displacing the cartridge in relative rotation, said method comprising:

a) at least partially filling said reservoir with a fluid containing a sample of nucleic acids to be analyzed;

b) distributing said fluid to the reaction chambers of said at least one circular cartridge, in which are located the primer and the reagents for carrying out an amplification reaction,

c) employing said means for relative rotational displacement to rotate said at least one circular cartridge to successively bring the contents of each reaction chamber to the at least two temperatures defined by the at least two distinct zones of said at least one heating plate wherein said means for relative rotational displacement comprises displacing said at least one circular cartridge while retaining in place said at least one heating plate,

wherein said nucleic acid is amplified.

10. The method according to claim 9, wherein said reaction chambers further comprise one or more probes.

11. The method according to claim 10, wherein said probes are labelled.

12. The method according to claim 9, wherein said reaction chambers contain a fluorescent intercalating agent.

13. The method according to claim 9, wherein said reaction chambers contain salts and deoxyribonucleoside triphosphates (dNTPs).

14. The method according to claim 12, wherein said fluorescent intercalating agent is deposited as a liquid and then dried.

15. The method according to claim 13, wherein said salts are deposited as a liquid and then dried.

16. The method of claim 9, comprising further exciting and measuring the fluorescence of the contents of the reaction chambers in each cycle, with optical means for fluorescence excitation/measurement.

17. The method of claim 9, said at least one heating plate is constituted by two or three distinct independent heating zones.

18. The method of claim 9, wherein distributing fluid to the reaction chambers is carried out by applying an underpressure inside said at least one circular cartridge, then re-establishing the pressure.

19. The method of claim 7, wherein said at least two or three distinct zones are the same size.

20. The method of claim 7, wherein said at least two or three distinct zones are a different size.

21. A method for amplifying a nucleic acid using a device comprising at least one circular cartridge having a plurality of reaction chambers and a reservoir, said reaction chambers being connected to the reservoir via channels, at least one heating plate having at least two distinct zones that can be heated to at least two different temperatures, wherein said distinct zones are sections of a disk or sections of a ring, and a means for relative rotational displacement between said at least one circular cartridge and said at least one heating plate, allowing a cyclic variation of the temperature of the reaction chambers wherein said means for relative rotational displacement is connected to said at least one circular cartridge for displacing the cartridge in relative rotation, said method comprising:

a) at least partially filling said reservoir with a fluid containing a sample of nucleic acids to be analyzed and reagents for carrying out an amplification reaction, with the exception of primers,

b) distributing said fluid to the reaction chambers of said at least one circular cartridge, in which are located the primers,

c) employing said means for relative rotational displacement to rotate said at least one circular cartridge to successively bring the contents of each reaction chamber to the at least two temperatures defined by the at least two distinct zones of said at least one heating plate wherein said means for relative rotational displacement comprises displacing said at least one circular cartridge while retaining in place said at least one heating plate,

wherein said nucleic acid is amplified.

22. A method for amplifying a nucleic acid using a device comprising at least one circular cartridge having a plurality of reaction chambers and a reservoir, said reaction chambers being connected to the reservoir via channels, at least one heating plate having at least two distinct zones that can be heated to at least two different temperatures, wherein said distinct zones are sections of a disk or sections of a ring, and a means for relative rotational displacement between said at least one circular cartridge and said at least one heating plate, allowing a cyclic variation of the temperature of the reaction chambers wherein said means for relative rotational displacement is a micromotor and a driver or an axle that is moved by means of the micromotor and the driver or axle is connected to said at least one circular cartridge for displacing the cartridge in relative rotation, said method comprising:

a) at least partially filling said reservoir with a fluid containing a sample of nucleic acids to be analyzed and reagents for carrying out an amplification reaction, with the exception of primers,

b) distributing said fluid to the reaction chambers of said at least one circular cartridge, in which are located the primers,

c) employing said means for relative rotational displacement to rotate said at least one circular cartridge to successively bring the contents of each reaction chamber to the at least two temperatures defined by the at least two distinct zones of said at least one heating,

wherein said nucleic acid is amplified.

23. A method for amplifying a nucleic acid using a device comprising a circular cartridge having a plurality of reaction chambers, and a reservoir, the reaction chambers being connected to the reservoir via channels, wherein at least a portion of the reaction chambers comprise primers;

and a heating plate having at least two distinct zones that can be heated to at least two different temperatures, the method comprising;

a) at least partially filling the reservoir with a fluid containing a sample of nucleic acids to be analyzed and reagents for carrying out an amplification reaction, with the exception of primers,

b) distributing the fluid to the reaction chambers comprising the primers therein,

c) rotating the circular cartridge to successively bring the contents of each reaction chamber to the at least two temperatures defined by the at least two distinct zones of the heating plate,

wherein the nucleic acid is amplified.

24. A method for amplifying a nucleic acid using a device comprising a circular cartridge having a plurality of reaction chambers and a reservoir, the reaction chambers being connected to the reservoir via channels, wherein at least a portion of the reaction chambers comprise primers and reagents for carrying out an amplification reaction;

and a heating plate having at least two distinct zones that can be heated to at least two different temperatures, the method comprising;

a) at least partially filling the reservoir with a fluid containing a sample of nucleic acids to be analyzed;

b) distributing the fluid to the reaction chambers comprising the primer and reagents therein,

c) rotating the circular cartridge to successively bring the contents of each reaction chamber to the at least two temperatures defined by the at least two distinct zones of the heating plate, while retaining in place the at least one heating plate,

wherein the nucleic acid is amplified.

25. A method for amplifying a nucleic acid using a device comprising a rotatable circular cartridge having a plurality of reaction chambers and a reservoir, the reaction chambers being connected to the reservoir via channels, wherein at least a portion of the reaction chambers comprise primers;

and a heating plate having at least two distinct zones that can be heated to at least two different temperatures, the method comprising;

a) at least partially filling the reservoir with a fluid containing a sample of nucleic acids to be analyzed and reagents for carrying out an amplification reaction, with the exception of primers;

b) distributing the fluid to the reaction chambers of the rotatable circular cartridge comprising the primers therein, wherein the rotatable circular cartridge is connected to a driver or axle, the driver or axle being movable by a micromotor;

c) moving, by a micromotor, the driver or axle connected to the rotatable circular cartridge, and rotating the rotatable circular cartridge to successively bring the contents of each reaction chamber to the at least two temperatures defined by the at least two distinct zones of the heating plate,

wherein the nucleic acid is amplified.

26. A method for amplifying a nucleic acid using a device comprising a rotatable circular cartridge having a plurality of reaction chambers and a reservoir, the reaction chambers being connected to the reservoir via channels, wherein at least a portion of the reaction chambers comprise primers and reagents for carrying out an amplification reaction;

and a heating plate having at least two distinct zones that can be heated to at least two different temperatures, the method comprising;

a) at least partially filling the reservoir with a fluid containing a sample of nucleic acids to be analyzed;

b) distributing the fluid to the reaction chambers of the rotatable circular cartridge comprising the primers and the reagents therein, wherein the rotatable circular cartridge is connected to a driver or axle, the driver or axle being movable by a micromotor;

c) moving, by a micromotor, the driver or axle connected to the rotatable circular cartridge, and rotating the rotatable circular cartridge to successively bring the contents of each reaction chamber to the at least two temperatures defined by the at least two distinct zones of the heating plate,

wherein the nucleic acid is amplified.

27. The method of claim 25, wherein the heating plate has at least three distinct zones that can be heated to at least three different temperatures, and c) comprises rotating the rotatable circular cartridge to successively bring the contents of each reaction chamber to the at least three temperatures defined by the at least three distinct zones of the heating plate.

28. The method of claim 23, wherein the reaction chambers comprise the primers and one or more probes, and b) comprises distributing the fluid to the reaction chambers comprising the primers and probes.