Method for analysing amplified nucleic acids

• US 8,551,737 B2
• Filed: 05/31/2007
• Issued: 10/08/2013
• 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: Gumbrecht, Walter, Paulicka, Peter, Stanzel, Manfred
• Primary Examiner(s): WILDER, CYNTHIA B
• Application Number:
  US12/308,552
• Publication Number:
  US 20100009864A1
• Time in Patent Office: 2,322 Days
• Field of Search: 435/91.2, 435/6.1
• US Class Current: 435/91.2
• 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 to produce an amplification crude product;

contacting the amplification crude product with an additive including monovalent cations and an Mg2+ ion binding agent to form a mixture of the amplification crude product and the additive;

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

wherein the additive is used to improve hybridization;

and hybridizing the nucleic acids in the amplification crude product to at least one probe oligonucleotide without having first purified the nucleic acids from the amplification crude product.

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 claim 1, wherein, prior to contacting the additive with the amplification crude product, a solvent is introduced into the second chamber.

5. The method as claimed in claim 4, wherein the additive is transferred in dissolved form from the second to the first chamber in order to contact the additive with the amplification crude product amplified.

6. The method as claimed in claim 1, wherein the amplification crude product is transferred from the first to the second chamber in order to contact the additive with the amplification crude product.

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

8. 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.

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

10. The method as claimed in claim 9, wherein the nucleic acids are labeled and the detection is effected using the label.

11. The method as claimed in claim 10, wherein the label is an optical label.

12. The method as claimed in claim 10, wherein the label is an enzymatic label.

13. The method as claimed in claim 12, wherein the label catalyzes an enzymatic reaction which is optically detectable.

14. The method as claimed in claim 12, wherein the label catalyzes an enzymatic reaction which is electrochemically detectable.

15. The method as claimed in claim 14, wherein the electrochemical detection involves a current measurement amplified by way of redox cycling.

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

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