Method for the detection of biomolecules based on the replication of bound nucleic acids

• US 20060183126A1
• Filed: 07/07/2004
• Published: 08/17/2006
• Est. Priority Date: 07/18/2003
• Status: Abandoned Application

• Current Assignee: QIAGEN North American Holdings Inc. (Qiagen NV)
• Original Assignee: Eppendorf AG
• Inventors: Bulow, Sven
• Application Number:
  US10/564,562
• Publication Number:
  US 20060183126A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2563/125 the label being enzymatic, i.e. proteins, and non proteins, such as nucleic acid with enzymatic activity
• CPC Class Codes: G01N 2458/10 Oligonucleotides as tagging agents for labelling antibodies

Abstract

A biomolecules detection method, where the biomolecules to be detected are coupled to a first substance which is part of a nucleic acid replicating device, the formed biomolecule/substance complexes being bound to the solid phase bound binding molecule specific to the particular biomolecule, and, if called for, the non-bound biomolecule/substance complexes being removed by washing, the bound biomolecule/substance complexes being incubated with high molecular weight nucleic acid molecules and mononucleotides of different species, of which at least the mononucleotides of one species are fitted with detectable markings, further being incubated with a second substance complementing the first substance coupled to the biomolecules into a functional, replicating device for high molecular weight nucleic acids that binds the high molecular weight nucleic acids, and with integration of marked mononucleotides generating replicas of the high molecular weight nucleic acid molecules that do not dissociate off it, where called for the dissolved high molecular weight nucleic acid molecules and mononucleotides which do not dissociate being removed by washing, the biomolecules to be detected being ascertained from the evidence of the marked replicas.

Claims

1. A method detecting biomolecules, where

(a) the biomolecules to be detected are coupled to a first substance which is part of a nucleic acid replication device,

(b) the formed biomolecules/substance complexes are bound to the solid phase bound binding molecules that are specific for the particular biomolecules,

(c) if called for the non-bound biomolecules/substance complexes are removed by washing,

(d) the bound biomolecules/substance complexes are incubated with high molecular weight nucleic acid molecules and mononucleotides of various species of which at least the nucleotides of one species are fitted with a detectable marking, further with a second substance which complements the first substance into a functional replicating device for high molecular weight nucleic acids, said device binding the high molecular weight nuclear acid molecules and under integration of marked mononucleotides generating replicas of the high molecular weight nucleic acid molecules and mononucleotides that do not dissociate off it,

(e) if called for, removing by washing the dissolved high molecular weight nucleic acid molecules and mononucleotides,

(f) and determining the biomolecules to be detected from the detection of the marked replicas.

4. Method for detecting biomolecules as claimed in claim 1, characterized in that the biomolecules to be detected are covalently coupled to the first substance.

6. Method for detecting biomolecules as claimed in claim 1, characterized in that the biomolecules to be detected are coupled by linker systems to the first substance.

9. Method for detecting biomolecules as claimed in claim 1, characterized in that the first substance contains the β

sub-unit of a DNA polymerase III and the second substance contains the remaining, required sub-units of a DNA polymerase III.

10. Method for detecting biomolecules as claimed in claim 1, characterized the first substance is one or several sub-units of a DNA polymerase III and the second substance contains β

sub-units of a DNA polymerase III and, where called for, further required sub-units of a DNA polymerase III.

11. Method for detecting biomolecules as claimed in claim 1, characterized in that the first substance contains β

sub-units of a DNA polymerase III and the second substance is a DNA polymerase I, the Klenow fragment of a DNA polymerase I, the Taq DNA polymerase or another DNA polymerase.

12. Method for detecting biomolecules as claimed in claim 1, characterized in that the first substance is a DNA polymerase I, the Klenow fragment of a DNA polymerase I, the Taq DNA polymerase or another DNA polymerase and the second substance contains β

subunits of DNA polmerase III.

2. A method detecting biomolecules, where

(a) immobilized biomolecules are incubated with connecting complexes consisting of binding molecules specific to the particular biomolecules and of a first substance that is part of a nucleic acid replication device,

(b) if called for removing by washing the non-bound connection complexes,

(c) incubating the formed biomolecule/complexes with high molecular weight nucleic acid molecules and mononucleotides of various species, at least the mononucleotides of one species being fitted with a detectable marking and with a second substance that complements the first substance coupled to the biomolecules into a functional, replicating device for high molecular weight nucleic acids, said device binding the high molecular weight nucleic acid molecules and generating under integration of marked mononucleotides replicas of the high molecular weight nucleic acid molecules which do not dissociate off it,

(d) if called for removing by washing the dissolved nucleic acid molecules and mononucleotides,

(e) and determining the biomolecules to be detected by detecting the marked replicas.

3. Method for detecting biomolecules as claimed in claim 2, characterized in that

(a) prior to incubation with the connection complexes, the biomolecules are immobilized by being bound to solid phase bound specific binding molecules,

(b) following incubation of the biomolecules with the connection complexes, the non-bound connection complexes are removed by washing with water if called for,

(c) prior to detecting the marked replicas, the dissolved high molecular weight nucleic acid molecules and mononucleotides are removed by washing if called for.

5. Method for detecting biomolecules as claimed in claim 2, characterized in that, in the connection complexes, the binding molecules are covalently coupled to the first substance.

7. Method for detecting biomolecules as claimed in claim 2, characterized in that, in the connection complexes, the binding molecules are coupled by means of linker system to the first substance.

44. Method for detecting biomolecules as claimed in claim 2, characterized in that the first substance contains the β

sub-unit of a DNA polymerase III and the second substance contains the remaining, required sub-units of a DNA polymerase III.

45. Method for detecting biomolecules as claimed in claim 2, characterized the first substance is one or several sub-units of a DNA polymerase III and the second substance contains β

sub-units of a DNA polymerase III and, where called for, further required sub-units of a DNA polymerase III.

46. Method for detecting biomolecules as claimed in claim 2, characterized in that the first substance contains β

sub-units of a DNA polymerase III and the second substance is a DNA polymerase I, the Klenow fragment of a DNA polymerase I, the Taq DNA polymerase or another DNA polymerase.

47. Method for detecting biomolecules as claimed in claim 2, characterized in that the first substance is a DNA polymerase I, the Klenow fragment of a DNA polymerase I, the Taq DNA polymerase or another DNA polymerase and the second substance contains β

sub-units of DNA polmerase III.

8. (canceled)

13-25. -25. (canceled)

26. A method for preparing a marker for the purpose of detecting biomolecules, where

a first substance which is part of a nucleic acid replicating device and which comprises a coupling element,

is incubated with high molecular weight nucleic acid molecules and mononucleotides of different species, of which at least the mononucleotides of one species are fitted with a detectable marking, also with a second substance that complements the first substance into a functional replicating device for high molecular weight nucleic acids, in such manner that

the device so formed binds the high molecular weight nucleic acid molecules, and, while integrating marked mononucleotides, does generate replicas of the high molecular weight nucleic acid molecules that do not dissociate off it.

27. Method for preparing a marker for the purpose of detecting biomolecules as claimed in claim 26, characterized in that the coupling element is a functional group able to covalently bind with molecules to be bound.

28. Method for preparing a marker for the purpose of detecting biomolecules as claimed in claim 26, characterized in that the coupling element is part of a linker system allowing to bind the molecules to be bound.

30. Method for preparing a marker for the purpose of detecting biomolecules as claimed in claim 26, characterized in that the first substance is connected by the coupling element to a binding molecule able to specifically binding a biomolecule.

31. Method for preparing a marker for the purpose of detecting biomolecules as claimed in claim 26, characterized in that the first substance is connected by the coupling element to a biomolecule.

32. Method for preparing a marker for the purpose of detecting biomolecules as claimed in claim 30, characterized in that the binding molecules are proteins, sugars, nucleic acids, antibodies, lectins, receptors or other specifically binding molecules.

33. Method for preparing a marker for the purpose of detecting biomolecules as claimed in claim 30, characterized in that the biomolecules to be detected are amino acids, proteins, sugars, nucleic acids, antibodies, lectins, lipids or receptors.

34. Method for preparing a marker for the purpose of detecting biomolecules as claimed in claim 26, characterized in that the first substance is the β

sub-unit of a DNA polymerase and the second substance contains the remaining, required sub-units of a DNA polymerase III.

35. Method for preparing a marker for the purpose of detecting biomolecules as claimed in claim 26, characterized in that the first substance is one or more sub-units of a DNA polymerase III and the second substance contains β

sub-units of a DNA polymerase and any further required sub-units of a DNA polymerase III.

36. Method for preparing a marker for the purpose of detecting biomolecules as claimed in claim 26, characterized in that the first substance contains β

sub-units of a DNA polymerase III and the second substance is a DNA polymerase I, the Klenow fragment of a DNA polymerase I, the Taq DNA polymerase or another DNA polymerase.

37. Method for preparing a marker for the purpose of detecting biomolecules as claimed in claim 26, characterized in that the first substance is a DNA polymerase I, the Klenow fragment of a DNA polymerase I, the Taq DNA polymerase or another DNA polymerase and the second substance contains β

sub-units of a DNA polymerase III.

43. A marker detecting biomolecules, characterized in that it is prepared by a method defined in claim 26.

29. (canceled)

38-42. -42. (canceled)