Integrated instrument performing synthesis and amplification, and a system and method thereof

• US 8,357,490 B2
• Filed: 07/22/2010
• Issued: 01/22/2013
• Est. Priority Date: 01/23/2008
• Status: Active Grant

• Current Assignee: Roche Diagnostics Operations Incorporated (Roche Holding AG)
• Original Assignee: Roche Diagnostics Operations Incorporated (Roche Holding AG)
• Inventors: Froehlich, Thomas, Gutekunst, Martin, Heindl, Dieter, Roesler, Angelika, Seibl, Rudolf
• Primary Examiner(s): Kim, Young J
• Application Number:
  US12/841,708
• Publication Number:
  US 20110177514A1
• Time in Patent Office: 915 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00286 Reactor vessels with top and bottom openings
• CPC Class Codes: B01J 2219/00308 interchangeably mounted in racks or blocks
• CPC Class Codes: B01J 2219/00315 Microtiter plates
• CPC Class Codes: B01J 2219/00364 Pipettes
• CPC Class Codes: B01J 2219/00423 using filtration, e.g. through porous frits
• CPC Class Codes: B01J 2219/00454 by chemical cleavage from the solid support
• CPC Class Codes: B01J 2219/00511 Walls of reactor vessels
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00639 the compounds being trapped in or bound to a porous medium
• CPC Class Codes: B01J 2219/00691 using robots
• CPC Class Codes: B01J 2219/00695 Synthesis control routines, e.g. using computer programs
• CPC Class Codes: B01J 2219/00722 Nucleotides
• 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: C40B 60/14 for creating libraries
• CPC Class Codes: G01N 2035/00326 Analysers with modular structure
• CPC Class Codes: G01N 2035/00356 Holding samples at elevated temperature (incubation)
• CPC Class Codes: G01N 35/028 having reaction cells in the form of microtitration plates
• CPC Class Codes: G01N 35/1002 : Reagent dispensers

Abstract

An integrated instrument for oligonucleotide synthesis and PCR, and a system and method thereof are disclosed. The integrated instrument is basically composed of two independent modules. The first module is a unit for chemical de novo synthesis of oligonucleotides such as oligonucleotide primers and/or oligonucleotide hybridization probes. The second module is a unit for performing an analytical polymerase chain reaction amplification in real time, i.e. a qPCR. The two modules are operatively linked to each other in such a way that a user can load a nucleic sample to be analyzed into the integrated instrument and perform a PCR reaction by programming the instrument without a previous external synthesis of oligonucleotide amplification primers.

Claims

1. An integrated instrument for performing de novo synthesis of a plurality of oligonucleotides and real time PCR amplification of a target nucleic acid, said integrated instrument comprising:

an oligonucleotide synthesizer unit for performing de novo synthesis of a plurality of oligonucleotides;

a real time PCR thermocycler unit for performing amplification of a target nucleic acid;

an interface unit operatively linking said synthesizer unit and said thermocycler unit; and

a central processing unit which controls said synthesizer unit, said thermocycler unit, and said interface unit, such that subsequent to de novo synthesis of said plurality of oligonucleotides said interface unit automatically transfers a plurality of said oligonucleotides to said thermocycler unit, whereby said thermocycler unit automatically performs said amplification of said target nucleic acid with said plurality of said oligonucleotides.

2. The instrument according to claim 1, wherein said interface unit comprises a pipetting robot.

3. The instrument according to claim 1, wherein said interface unit comprises a UV measurement device.

4. The instrument according to claim 3, wherein said UV measurement device performs a quality control measurement of said at least one of primers and probes prior to said amplification of said target DNA.

5. The instrument according to claim 1, wherein said interface unit further comprises a storage means for storing a plurality of multi-cavity disposables, which are microtiter plates or picotiter plates.

6. The instrument according to claim 1, wherein said interface unit comprises means for preparing at least a first PCR reaction mixture.

7. The instrument according to claim 6, wherein said interface unit further comprises a transport means for transporting said PCR reaction mixtures into said thermocycler unit.

8. A method of performing a real time PCR comprising:

(i) performing an oligonucleotide synthesis of a first primer and a second primer in an integrated instrument according to claim 1;

(ii) adding into a reaction vessel located in said integrated instrument

a) the target nucleic acids sample to be analyzed,

b) said first and second primer synthesized in step (i), said first and said second primers being added into the reaction vessel by the interface unit and

c) all other reagents necessary for a real time PCR of said target nucleic acid; and

(iii) amplifying and simultaneously monitoring amplification of the target DNA in said integrated instrument.

9. The instrument according to claim 1, wherein said synthesizer unit comprises means for holding at least one CPG column or an array of CPG columns, said array of CPG columns being in a microtiter plate format.

10. The instrument according to claim 9, wherein said interface unit comprises a pipetting robot.

11. The instrument according to claim 10, wherein said interface unit comprises a UV measurement device.

12. The instrument according to claim 11, wherein said interface unit further comprises a storage means for storing a plurality of multi-cavity disposables, which are microtiter plates or picotiter plates.

13. The instrument according to claim 12, wherein said interface unit comprises means for preparing at least a first PCR reaction mixture.

14. The instrument according to claim 13, wherein said interface unit further comprises a transport means for transporting said PCR reaction mixtures into said thermocycler unit.

15. The instrument according to claim 9, wherein said synthesizer unit further comprises a suction device connected to said microtiter plate format, wherein said suction device draws said oligonucleotides into a cavity of a disposable in said synthesizer unit.

16. The instrument according to claim 1, wherein said synthesizer unit further comprises a housing in which said de novo synthesis of said oligonucleotides is performed.

17. An integrated system for automated oligonucleotide synthesis and subsequent real time PCR of a target nucleic acid, comprising:

an oligonucleotide synthesizer unit including a solid support, a frit, suction means, a collection support, and a collection support closure means, the synthesizer unit adapted for de novo synthesis of a plurality of oligonucleotides utilizable in a real time PCR reaction;

a real time PCR unit including a thermocyler component, a detection component, and a plurality of sample cavities, the real time PCR unit adapted for performing a real time PCR reaction with a plurality of sample;

an interface unit including a pipetting robot and a flow-through UV measurement device, the interface unit mechanically linking the synthesizer unit and the real time PCR unit such that a plurality of the oligonucleotides synthesized by the synthesizer are automatically transferred from the synthesizer unit directly to the real time PCR unit for utilization in a real time PCR reaction; and

a central processing unit adapted for receiving and executing a plurality of computer readable instructions from a non-transient machine readable media, the central processing unit operatively linking the synthesizer unit, the real time PCR unit and the interface unit such that upon initiation of the plurality of computer readable instructions the synthesizer unit performs de novo synthesis of a plurality of oligonucleotides, the interface unit automatically transfers a plurality of the synthesized oligonucleotides to the real time PCR unit and the real time PCR unit automatically performs a real time PCR reaction of a plurality of sample.

18. The integrated system of claim 17, wherein the solid support comprises a microtiter plate having a plurality of reaction cavities, each of the plurality of reaction cavities having a bottom portion, a frit being disposed at each of the bottom portions.

19. The integrated system of claim 18, wherein the frit disposed at each of the bottom portions separates each reaction cavity from the collection support.