Method for biochemical analysis of dna and arrangement associated therewith

• US 20050164199A1
• Filed: 05/18/2003
• Published: 07/28/2005
• Est. Priority Date: 05/10/2002
• Status: Abandoned Application

• Current Assignee: Siemens AG
• Original Assignee: Siemens AG
• Inventors: Stanzel, Manfred, Sprinzl, Mathias
• Application Number:
  US10/514,019
• Publication Number:
  US 20050164199A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2527/127 the enzyme inhibitor or activator used
• CPC Class Codes: C12Q 2563/125 the label being enzymatic, i.e. proteins, and non proteins, such as nucleic acid with enzymatic activity

Abstract

A system with immobilized DNA is used in the fields of medicine, environmentology or criminology as an analytical tool in the analysis of nucleic acid. The immobilized DNA is provided with a biocatalytically active marker, such as an enzyme, an with an inhibitor substance which reversibly inhibits catalytic activity, or in addition to the immobilized biocatalytic marker, the immobilized DNA is provided with a substance which can reversibly inhibit the catalytic activity of the marker. Alternatively, an immobilized biocatalytically active marker can be provided with DNA as a scavenger which includes a substance as an inhibitor which can reversibly inhibit the activity of the marker. In another alternative, it is possible to use a complex including a molecule binding double-stranded DNA and a substance as an inhibitor which can reversibly inhibit the activity of the marker by interacting with the immobilized biocatalytically active marker. In all cases, the inhibitor or compound including an inhibitor and a molecule which can bind double-stranded DNA interacts with the biocatalytically active marker and defines the inactive state of the system. When the DNA, which is to be analyzed, is bonded, especially hybridized, to the DNA scavengers, the interaction between the biocatalytically active marker and the inhibitor is cancelled as a result of the formation of the double strand. The system is thus shifted from a first state into a second state defining the active state. A carrier with integrated microelectrodes is provided in the associated device, whereby the enzyme is either immobilized therein or is contained in a polymer network in the vicinity of the microelectrodes.

Claims

1. A method for detecting DNA with use being made of a system employing immobilized DNAs as an analytical tool, comprising:

fixing the enzyme in a stationary manner in the system which is used as an analytical tool and which contains a catcher DNA, an inhibitor and an enzyme, as biocatalytic label;

using the catcher DNA to permit, in a first inactive state of the system, the inhibitor and enzyme to interact;

forming, when the DNA to be analyzed is bound to the catcher DNA, a double strand due to the complementarity and at least one of abolishing and preventing the interaction between enzyme and inhibitor, wherein

in this way, the system is switched from the first inactive state to a second, active state; and

measuring the signals of at least one of the active and inactive state via an electrochemically detectable substance whose concentration changes due to the enzyme activity.

2. The method as claimed in claim 1, wherein the structure of the catcher DNA enables the inhibitor and biocatalyst to interact.

3. The method as claimed in claim 2, wherein immobilized DNA is used as catcher DNA and wherein the immobilized DNA is provided with the biocatalytically active label and, as inhibitor, a substance which is able, by interaction with the label, to inhibit its catalytic activity reversibly.

4. The method as claimed in claim 2, wherein an immobilized biocatalytic label is used and wherein a DNA, which is provided with a substance, as inhibitor, which is able, by interaction with the label, to inhibit its catalytic activity reversibly, is immobilized, as catcher, in the vicinity of the immobilized biocatalytic label.

5. The method as claimed in claim 2, wherein an immobilized biocatalytic label is used and wherein the immobilized biocatalytically active label is provided with a DNA, as catcher, which DNA, for its part, carries a substance, as inhibitor, which is able, by interaction with the label, to inhibit its activity reversibly.

6. The method as claimed in claim 1, wherein use is made of a complex composed of a double-stranded DNA-binding molecule and an inhibitor substance which is able, by interaction with the immobilized biocatalytically active label, to inhibit its activity reversibly, with the complex being bound, when the immobilized catcher DNA and the analyte DNA hybridize, to the resulting double strand and consequently no longer being available for inhibiting the biocatalytically active label.

7. The method as claimed in claim 1, wherein the inhibitor is a substance which binds reversibly to the enzyme and inhibits the enzymic activity.

8. The method as claimed in claim 1, wherein the DNA to be analyzed forms, by hybridization with the immobilized DNA, a double strand, i.e. a double helix, on account of the complementarity of the single-stranded DNAs.

9. The method as claimed in claim 1, wherein the first, inactive state and the second, active state of the system, and its change from_the first state to the second state, together constitute a switching function.

11. The method as claimed in claim 9, wherein the state of the switching function of the system is interrogated by determining the activity of the biocatalyst.

12. The method as claimed in claim 1, further-comprising:

using an enzyme;

forming an activatable switch from the enzyme; and

reading out the signal of the enzyme switch least one of optically and electrochemically.

13. The method as claimed in claim 12, wherein the enzyme switch is controlled by the catcher DNA and the DNA to be analyzed hybridizing under stringent conditions.

14. The method as claimed in claim 12, wherein a product which is at least one optically and electrochemically detectable is synthesized, and wherein the enzyme catalyzes the conversion of an undetectable substrate into a product at least one of optically and electrochemically detectable.

15. The method as claimed in claim 12, wherein the electrochemical read-out is effected at least one of amperometrically, potentiometrically and conductometrically.

16. The method as claimed in claim 12, wherein the measured values of the enzyme switch are output digitally and can be read off directly.

17. The method as claimed in claim 12, wherein the analyte DNA concentration is effected by correlation between the quantity of enzyme product released and the quantity of DNA to be analyzed which is hybridized.

18. The method as claimed in claim 17, wherein the enzyme switch is deactivated by interaction of the inhibitor with the enzyme.

19. The method as claimed in claim 18, wherein the enzyme is inactivated if the inhibitor is bound to the enzyme and in that the inhibitor is unavailable to the enzyme, because of the double strand, and the enzyme is active, when a double helix exists between the catcher DNA and the DNA to be analyzed.

28. The method as claimed in claim 1, wherein the partial double/single strandedness of the catcher DNA enables the inhibitor and biocatalyst to interact.

29. The method as claimed in claim 13, wherein a product which is at least one optically and electrochemically detectable is synthesized, and wherein the enzyme catalyzes the conversion of an undetectable substrate into a product at least one of optically and electrochemically detectable.

30. The method as claimed in claim 13, wherein the electrochemical read-out is effected at least one of amperometrically, potentiometrically and conductometrically.

31. The method as claimed in claim 14, wherein the electrochemical read-out is effected at least one of amperometrically, potentiometrically and conductometrically.

10. The method as claimed in 1, wherein the switching function of the system is effected by the DNA to be analyzed hybridizing to the immobilized DNA, as catcher.

20. An arrangement, comprising:

a support on which an enzyme is immobilized at a site;

a catcher DNA which is immobilized at the site;

an inhibitor which is covalently linked to the catcher DNA and a substrate, with, in a first state, the catcher DNA being folded by way of intramolecular hydrogen bonds such that the inhibitor inhibits the activity of the enzyme and the substrate is not transformed, and with, in a second state, the catcher DNA hybridizing with a DNA to be detected and thereby being folded such that the inhibitor is separated from the enzyme and the substrate is transformed, wherein the support includes integrated microelectrodes, with the enzyme being at least one of immobilized on the support, and being at least one of enclosed and immobilized in a polymer network in the vicinity of the microelectrodes, and wherein at least one of the product and the substrate of the enzymic reaction is electrochemically detectable at the microelectrodes.

21. The arrangement as claimed in claim 20, wherein the polymer network does not interfere with the activity of the enzyme and is permeable for the analyte DNA to be analyzed.

22. The arrangement as claimed in claim 20, wherein the enzyme is at least one of a phosphatase, esterase and protease.

23. The arrangement as claimed in claim 22, wherein the enzyme is composed of a polypeptide chain and wherein the polypeptide chain is immobilized without the enzyme losing any activity.

24. The arrangement as claimed in claim 23, wherein the enzyme is thermostable.

25. The arrangement as claimed in claim 24, wherein the enzyme can be produced by an expression system which comprises at least one recombinant plasmid.

32. The apparatus of claim 20, wherein at least one of the product and the substrate of the enzymic reaction is at least one of amperometrically, potentiometrically and conductometrically detectable at the microelectrodes.

26. (canceled)

27. (canceled)