Method for analysing amplified nucleic acids

• US 20100009864A1
• Filed: 05/31/2007
• Published: 01/14/2010
• Est. Priority Date: 06/19/2006
• Status: Active Grant

• Current Assignee: Boehringer Ingelheim Vetmedica GmbH (C.H. Boehringer Sohn AG & Co. KG)
• Original Assignee: Siemens AG
• Inventors: Paulicka, Peter, Gumbrecht, Walter, Stanzel, Manfred
• Granted Patent: US 8,551,737 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• 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: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

An example embodiment of the present invention discloses a method for analyzing nucleic acids in a microfluidic device. The method includes the following steps: a) nucleic acids are amplified in a first chamber in the microfluidic device; b) the amplified nucleic acids are brought into contact with an additive including: i) monovalent cations and ii) an Mg2+ ion-binding agent, the additive being provided in a second chamber in the microfluidic device; and c) the amplified nucleic acids are hybridized on at least one probe oligonucleotide.

Claims

1. A method for analyzing nucleic acids in a microfluidic device, comprising:

amplifying nucleic acids in a first chamber in the microfluidic device;

contacting the amplified nucleic acids with an additive including

monovalent cations and

an Mg²⁺ ion-binding agent,

wherein the additive is provided in a second chamber in the microfluidic device; and

hybridizing the amplified nucleic acids to at least one probe oligonucleotide.

2. The method as claimed in claim 1, wherein the monovalent cations are provided in the form of Na⁺ ions.

3. The method as claimed in claim 1, wherein the Mg²⁺ ion-binding agent is EDTA.

4. The method as claimed in claimed in claim 1, wherein the additive in the second chamber is provided as a dry reagent.

5. The method as claimed in claim 4, wherein, prior to contacting the additive with the amplified nucleic acids, a solvent is introduced into the second chamber.

6. The method as claimed in claim 5, wherein the additive is transferred in dissolved form from the second to the first chamber in order to contact the additive with the amplified nucleic acids.

7. The method as claimed claim 1, wherein the amplified nucleic acids are transferred from the first to the second chamber in order to contact the additive with the amplified nucleic acids.

8. The method as claimed in claim 7, wherein the amplified nucleic acids in solution as amplification crude product are transferred to the second chamber and then conducted as a mixture with the additive to the at least one probe oligonucleotide.

9. The method as claimed in claim 1, wherein the nucleic acids are amplified by way of PCR reaction.

10. The method as claimed in claim 1, wherein the at least one probe oligonucleotide is immobilized in the form of a microarray arrangement on a carrier.

11. The method as claimed in claim 1, wherein the amplified nucleic acids hybridized to the probe oligonucleotide are detected.

12. The method as claimed in claim 11, wherein the detection is effected using a label of the amplified nucleic acids.

13. The method as claimed in claim 12, wherein the label is an optical label.

14. The method as claimed in claim 12, wherein the label is an enzymatic label.

15. The method as claimed in claim 14, wherein the label catalyzes an enzymatic reaction which is optically detectable.

16. The method as claimed in claim 14, wherein the label catalyzes an enzymatic reaction which is electrochemically detectable.

17. The method as claimed in claim 16, wherein the electrochemical detection involves a current measurement amplified by way of redox cycling.

18. The method as claimed in claim 1, wherein the additive comprises a binder.

31. The method as claimed in claim 2, wherein the Mg²⁺ ion-binding agent is EDTA.

19. An arrangement for carrying out a method for analyzing nucleic acids in a microfluidic device, provided in the microfluidic device, comprising:

a first chamber designed for amplifying the nucleic acids, and

a second chamber, in which an additive, comprising

monovalent cations and

an Mg²⁺ ion-binding agent,

is kept in storage-stable fashion for later contact with the amplified nucleic acids to hybridize the amplified nucleic acids to at least one probe oligonucleotide, the second chamber being connectable in fluid communication via a connection to the first chamber.

20. The arrangement as claimed in claim 19, wherein the monovalent cations are provided in the form of Na⁺ ions.

21. The arrangement as claimed in claim 19, wherein the Mg²⁺ ion-binding agent is EDTA.

22. The arrangement as claimed in claim 19, further comprising means for transferring fluid selectively from the first to the second chamber.

23. The arrangement as claimed in claim 19, further comprising means for transferring fluid selectively from the second to the first chamber.

24. The arrangement as claimed in claim 19, wherein the additive is kept in the second chamber as a dry reagent.

25. The arrangement as claimed in claim 24, further comprising means for introducing a solvent into the second chamber.

26. The arrangement as claimed in claim 19, further comprising a microarray arrangement having probe oligonucleotides immobilized on a carrier.

27. The arrangement as claimed in claim 19, further comprising means for optically detecting hybridized nucleic acids.

28. The arrangement as claimed in claim 19, further comprising means for electrochemically detecting hybridized nucleic acids.

29. The arrangement as claimed in claim 28, wherein the electrochemical detection means are designed for measuring at least one of currents and potentials.

30. The arrangement as claimed in claim 19, wherein means for at least one of supplying heat and dissipating heat are assigned to the first chamber.

32. The arrangement as claimed in claim 20, wherein the Mg²⁺ ion-binding agent is EDTA.