Method and apparatus for detection or identification of DNA

• US 20050048501A1
• Filed: 09/02/2003
• Published: 03/03/2005
• Est. Priority Date: 09/02/2003
• Status: Abandoned Application

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Corn, Robert Marcus, Goodrich, Terry Thomas, Lee, Hye Jin
• Application Number:
  US10/653,365
• Publication Number:
  US 20050048501A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2565/628 being a surface plasmon resonance spectrometer

Abstract

DNA is detected using complementary RNA probes and an enzyme that attacks and hydrolyzes the RNA probes only when it has hybridized with target DNA. A low concentration of target DNA can therefore successively hydrolyze a larger amount of RNA whose loss may then be detected to indirectly determine the presence of the target DNA.

Claims

1. A method of detecting target DNA molecules comprising the steps of:

(a) introducing target DNA molecules into a test element holding RNA molecules complementary to the target DNA molecules;

(b) selectively destroying the RNA molecules that have hybridized with target DNA molecules; and

(c) detecting a loss of RNA molecules caused by the target DNA molecules successively hybridizing with different RNA molecules.

2. The method of claim 1 wherein step (b) introduces an enzyme into the test cell to destroy the hybridized RNA.

3. The method of claim 2 wherein the enzyme is RNase H.

4. The method of claim 1 wherein steps (a) and (b) are performed at the same time.

5. The method of claim 4 wherein step (b) introduces an enzyme into the test cell to destroy the hybridized RNA and wherein the target DNA and enzyme are mixed together in a solution introduced into the test cell.

6. The method of claim 1 wherein the test element is a surface to which the RNA molecules are attached.

7. The method of claim 6 wherein the surface is selected from the group consisting of:

gold, diamond, glass, and silicon.

8. The method of claim 6 wherein the test element is a surface plasmon resonance (SPR) test cell to which the RNA molecules are attached, and step (c) is performed with an SPR imaging instrument detecting the loss of RNA molecules from the test cell.

9. The method of claim 8 wherein step (c) includes the steps of obtaining a baseline refraction measurement of the surface of the SPR test cell before step (b) and comparing the baseline refraction measurement to a refraction measurement of the surface of the SPR test cell after step (b).

10. The method of claim 8 wherein the SPR instrument produces an image of the SPR test cell and including the step before step (a) of attaching different RNA molecules to the surface in a predetermined pattern discernable in the image.

11. The method of claim 10 wherein the predetermined pattern is rotationally asymmetric.

12. The method of claim 1 wherein the SPR surface further includes control RNA that is not complementary to the target DNA and including the step of detecting the presence of the control RNA.

13. The method of claim 1 wherein the concentration of target DNA is less than 10 nM.

14. The method of claim 1 wherein the RNA molecules are tagged with a fluorescent material and wherein step (c) is performed with a fluorescence detection apparatus.

15. The method of claim 14 wherein step (c) includes the steps of obtaining a baseline fluorescence measurement of the surface of the RNA molecules of the test cell before step (b) and comparing the baseline fluorescence measurement to a fluorescence measurement of the RNA molecules of the test cell after step (b).

16. The method of claim 14 wherein the fluorescence detection apparatus produces an image of the test cell and including the step before step (a) of attaching the RNA molecules to a surface of the test cell in a predetermined pattern discernable in the image.

17. The method of claim 1 wherein the RNA molecules are tagged with a radioactive material and wherein step (c) is performed with a radiation detector.

18. The method of claim 14 wherein the radiation detector produces an image of the test cell and including the step before step (a) of attaching the RNA molecules to a surface of the test cell in a predetermined pattern discernable in the image.

19. The method of claim 16 wherein the predetermined pattern is rotationally asymmetric.

21. The apparatus of claim 18 wherein the means for destroying RNA molecules is an enzyme that selectively hydrolyses hybridized RNA.

22. The apparatus of claim 21 wherein the enzyme is RNase H.

23. The apparatus of claim 22 wherein the enzyme is mixed in solution with the target DNA for introduction through the port.

20. An apparatus for detecting target DNA molecules comprising:

a test element holding RNA molecules complementary to the target DNA molecules;

a port for introducing target DNA molecules into a test element;

a means for destroying the RNA molecules in the test cell selective to RNA molecules that have hybridized with target DNA molecules; and

a detector sensitive to a loss of RNA molecules caused the target DNA molecules successively hybridizing with different RNA molecules.

24. The apparatus of claim 20 wherein the test element is a surface to which the RNA molecules are attached.

25. The apparatus of claim 24 wherein the surface is selected from the group consisting of:

gold, diamond, and silicon.

26. The apparatus of claim 24 wherein the test element is a surface plasmon resonance (SPR) test cell to which the RNA molecules are attached and the detector is an SPR imaging instrument detecting the loss of RNA molecules from the test cell.

27. The apparatus of claim 26 wherein the SPR instrument produces an image of the SPR test cell and wherein the RNA molecules include different RNA molecules attached to the surface in a predetermined pattern discernable in the SPR image.

28. The apparatus of claim 27 wherein the predetermined pattern is rotationally asymmetric.

29. The apparatus of claim 20 wherein the SPR surface further includes control RNA that is not complementary to the target DNA.

30. The apparatus of claim 20 wherein the RNA molecules are tagged with a fluorescent material and the detector is a fluorescence detection apparatus.

31. The apparatus of claim 30 wherein the fluorescence detection apparatus produces an image of the test cell and wherein the RNA molecules are attached to a surface of the test cell in a predetermined pattern discernable in the image.

32. The apparatus of claim 31 wherein the predetermined pattern is rotationally asymmetric.