Method for labeling or treating a biological sample containing biological molecules of interest, in particular nucleic acids

• US 8,637,322 B2
• Filed: 05/31/2006
• Issued: 01/28/2014
• Est. Priority Date: 06/01/2005
• Status: Active Grant

• Current Assignee: Biomerieux B.V. (Compagnie Merieux Alliance SAS)
• Original Assignee: Biomerieux B.V. (Compagnie Merieux Alliance SAS)
• Inventors: Bernal-Mendez, Eloy, Laayoun, Ali, Menou, Lionel
• Primary Examiner(s): Menon, Krishnan S
• Assistant Examiner(s): Fritchman, Rebecca M
• Application Number:
  US11/919,668
• Publication Number:
  US 20090215188A1
• Time in Patent Office: 2,799 Days
• Field of Search: 436/94, 436/43, 436/903, 536/25.32, 536/32, 435/6.1, 435/91.2
• US Class Current: 436/94
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence
• CPC Class Codes: Y10T 436/11 Automated chemical analysis
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention relates to a method for labeling biological molecules of interest contained in a biological sample, consisting in:

• • a) providing a reaction vessel,
  • b) immobilizing capture molecules, which are capable of binding a label of the biological molecules of interest, on a solid support of the vessel,
  • c) introducing the biological sample but also at least one label of the biological molecules of interest into said vessel and optionally any ingredient required for labeling or prelabeling the molecules of interest,
  • d) incubating the vessel content and immobilizing the label which is not reacted with the molecules of interest by binding to the capture molecules, and
  • e) using the labeled molecules of interest for subsequent steps.

A method for treating a biological sample is also disclosed.

Said invention is preferably used in a manual or automated method for purifying nucleic acids.

Claims

1. A method for labeling nucleic acids, the method comprising:

introducing the nucleic acids and labels or labeling precursors into a reaction chamber, the labels or labeling precursors comprising a diazo function and the reaction chamber having immobilized capture molecules capable of binding the labels or labeling precursors at least partially on an inner surface of, or on a solid support in, the reaction chamber;

reacting the nucleic acids with the labels or labeling precursors to label or pre-label, respectively, the nucleic acids by reacting the diazo function with phosphate groups of the nucleic acids; and

covalently bonding unreacted labels or labeling precursors to the capture molecules.

5. The method as claimed in claim 1, wherein the inside surface of, or the solid support in, the reaction chamber comprises carboxylic and/or sulfonic functions.

6. The method as claimed in claim 1, wherein the nucleic acids are selected from the group consisting of DNA, RNA, and DNA-RNA chimeric polymers, which optionally contain at least one nucleotide thiophosphate, an LNA, a 2′

-O-Me and/or a methylphosphonate derivative.

9. The method as claimed in claim 1, wherein the capture molecules are present in excess relative to the labels or labeling precursors, and the labels or labeling precursors are present in excess relative to the nucleic acids to be labeled or pre-labeled.

10. The method as claimed in claim 1, wherein the capture molecules are present in excess relative to the unreacted labels or labeling precursors, and the labels or labeling precursors are present in excess relative to the nucleic acids to be labeled or pre-labeled.

11. The method as claimed in claim 1, wherein the capture molecules comprise carboxylic and/or sulfonic functions, and the unreacted labels or labeling precursors are covalently bonded to the capture molecules by reacting the diazo function with the carboxylic and/or sulfonic functions.

13. The method as claimed in claim 1, wherein the reaction chamber is a single reaction chamber in which the nucleic acids are reacted with the labels or labeling precursors and the unreacted labels or labeling precursors are covalently bonded to the capture molecules.

2. A method for treating a biological sample containing nucleic acids of interest, the method comprising:

introducing the biological sample and labels or labeling precursors into a reaction chamber, the labels or labeling precursors comprising a diazo function and the reaction chamber having immobilized capture molecules capable of binding the labels or labeling precursors at least partially on an inner surface of, or on a solid support in, the reaction chamber;

reacting the nucleic acids of interest with the labels or labeling precursors to label or pre-label, respectively, the nucleic acids by reacting the diazo function with phosphate groups of the nucleic acids; and

covalently bonding unreacted labels or labeling precursors to the capture molecules.

3. The method as claimed in claim 2, wherein, prior to introducing the biological sample into the reaction chamber, the method further comprises at least one of the following steps:

transferring the biological sample to the reaction chamber from an upstream reaction chamber,

lysing a complex biological material in order to make the nucleic acids of interest accessible and/or detectable,

capturing or isolating the nucleic acids of interest, and

treating the nucleic acids of interest in order to make their detection possible or to enhance their detection.

4. The method as claimed in claim 2, wherein the method further comprises at least one subsequent step below:

transferring the labeled or pre-labeled nucleic acids of interest to another reaction chamber downstream,

labeling the pre-labeled nucleic acids of interest to obtain labeled nucleic acids,

purifying the labeled or pre-labeled nucleic acids of interest, and

detecting nucleic acids of interest that are hybridized to capture probes.

7. The method as claimed in claim 3, wherein, prior to introducing the biological sample into the reaction chamber, the method further comprises treating the biological sample according to at least one of the following steps:

extracting nucleic acids from the complex biological material,

performing specific amplification of the nucleic acids of interest,

fragmenting the nucleic acids of interest or amplicons, and

transcribing or reverse transcribing the nucleic acids of interest, without amplification.

8. The method as claimed in claim 4, wherein the method further comprises at least one subsequent step below:

transcribing or reverse transcribing the nucleic acids of interest, without amplification and

detecting by homogeneous-phase detection the labeled or pre-labeled nucleic acids, with or without the use of detection probes.

12. The method as claimed in claim 2, wherein the capture molecules comprise carboxylic and/or sulfonic functions, and the unreacted labels or labeling precursors are covalently bonded to the capture molecules by reacting the diazo function with the carboxylic and/or sulfonic functions.

14. The method as claimed in claim 2, wherein the reaction chamber is a single reaction chamber in which the nucleic acids of interest are reacted with the labels or labeling precursors and the unreacted labels or labeling precursors are covalently bonded to the capture molecules.