Method for analyzing nucleic acid reactions

• US 6,607,888 B2
• Filed: 04/19/2001
• Issued: 08/19/2003
• Est. Priority Date: 10/20/1998
• Status: Expired due to Term

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Wu, Tian, Schwartz, David C.
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): KIM, YOUNG J
• Application Number:
  US09/838,497
• Publication Number:
  US 20020061523A1
• Time in Patent Office: 852 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2537/1376 Displacement by an enzyme
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/601 being a microscope, e.g. atomic force microscopy [AFM]
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

Disclosed are methods for analyzing reactions involving nucleic acids. The invention utilizes nucleic acids immobilized on a defined substrate in such a manner that the nucleic acid can participate in enzymatic and chemical reactions. These reactions are carried out in the presence of labeled reagents, thereby enabling the progress of the reactions to be analyzed using various techniques, such a fluorescent microscopy. The invention is particularly well suited for investigating transcription phenomena and generating genome-wide maps based upon reactions of individual nucleic acid molecules.

Claims

1. A method of analyzing enzymatic and chemical reactions of nucleic acids, the method comprising:

(a) elongating and fixing onto a surface of a substrate a plurality of nucleic acid molecules in such a fashion that each individual nucleic acid molecule is fixed along its length onto the surface of the substrate with a small degree of relaxation so that the nucleic acid molecules are individually analyzable and accessible for enzymatic and chemical reactions;

then

(b) subjecting the elongated and fixed nucleic acid of step (a) to an enzymatic or chemical reaction in the presence of a labeled reagent that generates signals correlating to the enzymatic or chemical reaction; and

then

(c) acquiring and compiling the signals generated by the labeled reagent, whereby the enzymatic or chemical reaction of step (b) is analyzed.

2. The method of claim 1, wherein in step (a), DNA is elongated and fixed to the substrate.

3. The method of claim 1, wherein in step (a), RNA is elongated and fixed to the substrate.

4. The method of claim 1, wherein in step (a), the nucleic acid is fixed to a glass substrate.

5. The method of claim 1, wherein in step (a), the nucleic acid is fixed to a glass substrate derivatized by a coating of a cationic substance disposed thereon.

6. The method of claim 1, wherein in step (a), the nucleic acid is fixed to a glass substrate derivatized by a coating selected from the group consisting of poly(lysine), 3-aminopropyltriethoxysilane, N-(trimethoxysilyl)propyl-N,N,N-trimethylammonium chloride, and vinyltrimethoxysilane.

7. The method of claim 1, wherein in step (b), the fixed nucleic acids are subjected to an enzymatic or chemical reaction in the presence of fluorescently-labeled reagent.

8. The method of claim 7, wherein in step (c), the signals generated by the fluorescently-labeled reagent are acquired using a camera and a microscope.

9. The method of claim 8, further comprising, after step (c):

(d) processing the compiled signals using Bayesian estimation.

10. A method of analyzing enzymatic and chemical reactions of nucleic acids, the method comprising:

(a) elongating and fixing onto a surface of a substrate a plurality of nucleic acid molecules in such a fashion that each individual nucleic acid molecule is fixed along its length onto the surface of the substrate with a small degree of relaxation so that the nucleic acid molecules are individually analyzable and accessible for enzymatic and chemical reactions;

then

(b) subjecting the elongated and fixed nucleic acids of step (a) to an enzymatic transcription reaction in the presence of a labeled reagent that generates signals correlating to the enzymatic transcription reaction; and

then

(c) acquiring and compiling the signals generated by the labeled reagent, whereby the enzymatic transcription reaction of step (b) is analyzed.

11. The method of claim 10, wherein in step (b), the fixed nucleic acids are subjected to an enzymatic transcription reaction in the presence of labeled reagent that comprises fluorescently-labeled NTP.

12. The method of claim 11, wherein in step (c), the signals generated by the fluorescently-labeled NTP are acquired using a camera and a microscope.

13. The method of claim 10, further comprising, after step (c):

(d) processing the compiled signals using Bayesian estimation.

14. The method of claim 10, wherein in step (a), genomic DNA molecules are elongated and fixed onto the surface of the substrate.

15. The method of claim 10, further comprising after step (c):

(d) manipulating the acquired and compiled signals generated by the labeled reagent into an image, and then observing an individual elongated nucleic acid molecule for appearance of complexes corresponding to transcription events in the individual nucleic acid molecule.

16. The method of claim 10, further comprising, after the transcription reaction of step (b) and prior to step (c), cleaving the nucleic acid molecules with a restriction enzyme to generate restriction fragments.

17. The method of claim 16, further comprising after step (c):

(d) manipulating the acquired and compiled signals generated by the labeled reagent into an image, and then;

(i) observing an individual elongated nucleic acid molecule for appearance of complexes corresponding to transcription events in the individual nucleic acid molecule; and

(ii) observing the individual elongated nucleic acid molecule of step (d) (i) for appearance of gaps corresponding to cleavage sites between restriction fragments.

18. The method of claim 17, further comprising reiteratively repeating steps (d), (d) (i), and (d) (ii) on additional individual elongated nucleic acid molecules, to thereby generate additional images, and then:

(e) compiling an ordered map correlating transcription event sites and restriction enzyme cleavage sites based upon the images.

19. The method of claim 18, wherein in step (a), genomic DNA molecules are elongated and fixed onto the surface of the substrate, and in step (e), compiling an ordered, genome-wide map correlating transcription event sites and restriction enzyme cleavage sites.

20. The method of claim 19, further comprising:

(f) comparing the map of step (e) to known genomic sequences, whereby it can be determined from where in a genome a single nucleic acid molecule originated.

21. A method of analyzing enzymatic and chemical reactions of nucleic acids, the method comprising:

(a) elongating and fixing onto a surface of a substrate a plurality of nucleic acid molecules in such a fashion that each individual nucleic acid molecule is fixed along its length onto the surface of the substrate with a small degree of relaxation so that the nucleic acid molecules are individually analyzable and accessible for enzymatic and chemical reactions;

then

(b) subjecting the elongated and fixed nucleic acid of step (a) to a transcription reaction followed by a restriction reaction in the presence of a labeled reagent that generates signals correlating to the transcription reaction and the restriction reaction, respectively;

then

(c) acquiring and compiling the signals generated by the labeled reagent;

then

(d) manipulating the acquired and compiled signals generated by the labeled reagent into an image, then;

(i) observing an individual elongated nucleic acid molecule for appearance of complexes corresponding to transcription events in the individual nucleic acid molecule; and

(ii) observing the individual elongated nucleic acid molecule of step (d) (i) for appearance of gaps corresponding to cleavage sites between restriction fragments;

then

(e) reiteratively repeating steps (d), (d) (i), and (d) (ii) on additional individual elongated nucleic acid molecules, to thereby generate additional images, and then

(f) compiling an ordered map correlating transcription event sites and restriction enzyme cleavage sites based upon the images.

22. The method of claim 21, further comprising, after step (f), comparing the map of step (f) to known genomic sequences, whereby it can be determined from where within a genome a single nucleic acid molecule originated.

23. A method of analyzing enzymatic and chemical reactions of nucleic acids, the method comprising:

(a) subjecting nucleic acid molecules to an enzymatic or chemical reaction in the presence of a labeled reagent that generates signals correlating to the enzymatic or chemical reaction, thereby generating nucleic acid reaction products; and

then

(b) elongating and fixing onto a surface of a substrate a plurality of the nucleic acid reaction products of step (a) in such a fashion that each individual nucleic acid molecule is fixed along its length onto the surface of the substrate with a small degree of relaxation so that the nucleic acid molecules are individually analyzable and accessible for further enzymatic and chemical reactions;

then

(c) acquiring and compiling the signals generated by the labeled reagent, whereby the enzymatic or chemical reaction of step (a) is analyzed.

24. The method of claim 23, wherein in step (b), DNA reaction products are elongated and fixed to the substrate.

25. The method of claim 23, wherein in step (b) RNA reaction products are elongated and fixed to the substrate.

26. The method of claim 23, wherein in step (b), the nucleic acid reaction products are fixed to a glass substrate.

27. The method of claim 23, wherein in step (b), the nucleic acid reaction products are fixed to a glass substrate derivatized by a coating of a cationic substance disposed thereon.

28. The method of claim 23, wherein in step (b), the nucleic acid reaction products are fixed to a glass substrate derivatized by a coating selected from the group consisting of poly(lysine), 3-aminopropyl-triethoxysilane, N-(trimethoxysilyl) propyl-N,N,N-trimethylammonium chloride, and vinyltrimethoxysilane.

29. The method of claim 23, wherein in step (a), the fixed nucleic acids are subjected to an enzymatic or chemical reaction in the presence of a fluorescently-labeled reagent.

30. The method of claim 29, wherein in step (c), the signals generated by the fluorescently-labeled reagent are acquired using a camera and a microscope, and the signals are compiled using a programmable computer.

31. The method of claim 30, further comprising, after step (c):

(d) processing the compiled signals using Bayesian estimation.

32. The method of claim 23, wherein in step (a), the nucleic acid molecules are subjected to an enzymatic transcription reaction.

33. The method of claim 32, wherein in step (a), the nucleic acid molecules are subjected to an enzymatic transcription reaction in the presence of labeled reagent that comprises fluorescently-labeled NTP.

34. The method of claim 33, wherein in step (c), the signals generated by the fluorescently-labeled NTP are acquired using a camera and a microscope.

35. The method of claim 32, further comprising, after step (c):

(d) processing the compiled signals using Bayesian estimation.

36. The method of claim 32, further comprising after step (c):

(d) manipulating the acquired and compiled signals generated by the labeled reagent into an image, and then observing an individual elongated nucleic acid molecule for appearance of complexes corresponding to transcription events in the individual nucleic acid molecule.

37. The method of claim 32, further comprising, after the transcription reaction of step (a) and prior to step (b), cleaving the nucleic acid molecules with a restriction enzyme to generate restriction fragments.

38. The method of claim 37, further comprising after step (c):

(d) manipulating the acquired and compiled signals generated by the labeled reagent into an image, and then;

(i) observing an individual elongated nucleic acid molecule for appearance of complexes corresponding to transcription events in the individual nucleic acid molecule; and

(ii) observing the individual elongated nucleic acid molecule of step (d) (i) for appearance of gaps corresponding to cleavage sites between restriction fragments.

39. The method of claim 38, further comprising reiteratively repeating steps (d), (d) (i), and (d) (ii) on additional individual elongated nucleic acid molecules, to thereby generate additional images, and then:

(e) compiling an ordered map correlating transcription event sites and restriction enzyme cleavage sites based upon the images.

40. The method of claim 39, wherein in step (a), genomic DNA molecules are used, and in step (e), compiling an ordered, genome-wide map correlating transcription event sites and restriction enzyme cleavage sites.

41. The method of claim 40, further comprising:

(f) comparing the map of step (e) to known genomic sequences, whereby it can be determined from where in a genome a single nucleic acid molecule originated.

42. A method of analyzing enzymatic and chemical reactions of nucleic acids, the method comprising:

(a) subjecting a nucleic acid to a transcription reaction followed by a restriction reaction in the presence of a labeled reagent that generates signals correlating to the transcription and the restriction reaction, respectively, thereby generating nucleic acid reaction products;

then

(a) elongating and fixing onto a surface of a substrate a plurality of nucleic acid reaction products of step (a) in such a fashion that each individual nucleic acid molecule is fixed along its length onto the surface of the substrate with a small degree of relaxation so that the nucleic acid molecules are individually analyzable and accessible for enzymatic and chemical reactions;

then

(c) acquiring and compiling the signals generated by the labeled reagent;

then

(d) manipulating the acquired and compiled signals generated by the labeled reagent into an image, then;

(i) observing an individual elongated nucleic acid molecule for appearance of complexes corresponding to transcription events in the individual nucleic acid molecule; and

(ii) observing the individual elongated nucleic acid molecule of step (d) (i) for appearance of gaps corresponding to cleavage sites between restriction fragments;

then

(e) reiteratively repeating steps (d), (d) (i), and (d) (ii) on additional individual elongated nucleic acid molecules, to thereby generate additional images, and then

(f) compiling an ordered map correlating transcription event sites and restriction enzyme cleavage sites based upon the images.