Methods and apparati using single polymer analysis

• US 7,371,520 B2
• Filed: 05/28/2003
• Issued: 05/13/2008
• Est. Priority Date: 05/28/2002
• Status: Expired due to Fees

• Current Assignee: U.S. Genomics, Inc (Toxic Reports LLC)
• Original Assignee: U.S. Genomics, Inc (Toxic Reports LLC)
• Inventors: Gullans, Steve R., Kundu, Bijit, Zhao, Xiaojian (David), Chan, Eugene Y., Fuchs, Martin, Randall, Jeffrey D., Kesty, Jessica
• Primary Examiner(s): Whisenant; Ethan
• Application Number:
  US10/448,264
• Publication Number:
  US 20040009612A1
• Time in Patent Office: 1,812 Days
• Field of Search: 435/6, 536/23.1, 536/24.3
• US Class Current: 435/6.19
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/601 being a microscope, e.g. atomic force microscopy [AFM]
• CPC Class Codes: C12Q 2565/619 being a video camera
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

The invention relates to methods for analyzing and characterizing single polymers such as nucleic acid molecules. In preferred embodiments, the single molecules are analyzed using single molecule detection and analysis systems.

Claims

1. A method for analyzing a single nucleic acid molecule comprising

exposing a single nucleic acid molecule to at least two distinguishable detectable labels present on different unit specific markers for a time sufficient to allow the detectable labels to bind to the single nucleic acid molecule, and

passing the single nucleic acid molecule in flow through a diffraction-limited detection spot, thereby analyzing the single nucleic acid molecule for a coincident event using a single molecule detection system by detecting two separate and distinguishable signals from the at least two distinguishable detectable labels,

wherein the single nucleic acid molecule is present at a concentration of 400 fM or greater, the coincidence event indicates that the at least two distinguishable detectable labels are bound to the single nucleic acid molecule, the coincident event is a color coincident event, and the method analyzes 20-20,000 single nucleic acid molecules per minute.

2. The method of claim 1, wherein the single nucleic acid molecule is denatured to a single stranded form.

3. The method of claim 1, wherein the single nucleic acid molecule is an RNA.

4. The method of claim 1, wherein the single nucleic acid molecule is linearized or stretched prior to analysis.

7. The method of claim 1, further comprising a column purification step.

8. The method of claim 1, wherein the single nucleic acid molecule is present in a nanoliter volume.

9. The method of claim 1, wherein the single nucleic acid molecule is present in at a frequency of 1 in 1,000,000 molecules in an RNA sample.

10. The method of claim 1, wherein each of the at least two distinguishable detectable labels is present on a unit specific marker that is a DNA, RNA, PNA, LNA or a combination thereof.

11. The method of claim 1, further comprising exposing the nucleic acid molecule to a ligase prior to analysis using the single molecule detection system.

12. The method of claim 1, wherein unbound detectable labels are not removed prior to analysis using the single molecule detection system.

13. The method of claim 1, wherein the detectable labels are provided as molecular beacon probes.

14. The method of claim 1, wherein at least one detectable label is attached to a nucleic acid molecule hybridized to a universal linker attached to a unit specific marker.

5. A method for analyzing a single nucleic acid molecule comprising exposing a single nucleic acid molecule to at least two distinguishable detectable labels for a time sufficient to allow the detectable labels to bind to the single nucleic acid molecule,

exposing the single nucleic acid molecule and detectable labels to a chemical or enzymatic single stranded cleavage reaction, and

then analyzing the single nucleic acid molecule for a coincident event using a single molecule detection system by detecting two separate and distinguishable signals from the at least two distinguishable detectable labels,

wherein the coincidence event indicates that the at least two distinguishable detectable labels are bound to the single nucleic acid molecule, and the coincident event is a color coincident event.

6. The method of claim 5, wherein the enzymatic single stranded cleavage reaction uses a single stranded RNA nuclease, a single stranded DNA nuclease, or a combination thereof.

15. A method for analyzing a single nucleic acid molecule comprising

exposing a single nucleic acid molecule to at least two distinguishable detectable labels for a time sufficient to allow the detectable labels to bind to the single nucleic acid molecule wherein the at least two distinguishable detectable labels are present on the same unit specific marker,

analyzing the single nucleic acid molecule for a coincident event using a single molecule detection system wherein the coincidence event indicates that the at least two distinguishable detectable labels are bound to the single nucleic acid molecule, and

exposing the single nucleic acid molecule to a third detectable label that binds specifically to a mismatch between the single nucleic acid molecule and the unit specific marker, wherein a coincident event between the first, second and third detectable labels is indicative of a mismatch.

25. The method of claim 5, 15, 16, 17, 18 or 23, wherein the single nucleic acid molecule is passed in flow through a diffraction-limited detection spot and is thereby analyzed.

26. The method of claim 5, 15, 16, 17, 18 or 23, wherein the single nucleic acid molecule is present at a concentration of 400 fM or greater.

27. The method of claim 5, 15, 16, 17, 18 or 23, wherein the method analyzes 20-20,000 single nucleic acid molecules per minute.

28. The method of claim 15, 16, 17 or 18, wherein analyzing the single nucleic acid molecule comprises detecting two separate and distinguishable signals from the at least two distinguishable detectable labels.

16. A method for analyzing a single nucleic acid molecule comprising

exposing a single nucleic acid molecule to at least two distinguishable detectable labels for a time sufficient to allow the detectable labels to bind to the single nucleic acid molecule,

analyzing the single nucleic acid molecule for a coincident event using a single molecule detection system, and

exposing the single nucleic acid molecule and detectable labels to an enzymatic single stranded cleavage reaction that uses RNase I prior to analyzing the single nucleic acid molecule,

wherein the coincidence event indicates that the at least two distinguishable detectable labels are bound to the single nucleic acid molecule.

17. A method for analyzing a single nucleic acid molecule comprising

exposing a single nucleic acid molecule to at least two distinguishable detectable labels for a time sufficient to allow the detectable labels to bind to the single nucleic acid molecule,

analyzing the single nucleic acid molecule for a coincident event using a single molecule detection system, and

exposing the single nucleic acid molecule and detectable labels to an enzymatic single stranded cleavage reaction that uses S1 nuclease prior to analyzing the single nucleic acid molecule,

wherein the coincidence event indicates that the at least two distinguishable detectable labels are bound to the single nucleic acid molecule.

18. A method for analyzing a single nucleic acid molecule comprising

exposing a single nucleic acid molecule to at least two distinguishable detectable labels for a time sufficient to allow the detectable labels to bind to the single nucleic acid molecule in the presence of a polymerase, a labeled unit specific marker and labeled nucleotides, provided the unit specific marker and nucleotides are differentially labeled with the at least two distinguishable detectable labels, and

analyzing the single nucleic acid molecule for a coincident event using a single molecule detection system,

wherein the coincidence event indicates that the at least two distinguishable detectable labels are bound to the single nucleic acid molecule.

19. The method of claim 18, wherein a new nucleic acid molecule is formed starting at the unit specific marker and is complementary to the single nucleic acid molecule.

20. The method of claim 19, wherein the new nucleic acid molecule has a signal intensity proportional to its length, and wherein the method is a method of determining integrity of the single nucleic acid molecule.

21. The method of claim 18, wherein the polymerase is a DNA polymerase.

22. The method of claim 19, wherein the polymerase is a reverse transcriptase.

23. A method for analyzing a single nucleic acid molecule comprising

exposing a single nucleic acid molecule to at least two distinguishable detectable labels for a time sufficient to allow the detectable labels to bind to the single nucleic acid molecule in the presence of a polymerase, a labeled unit specific marker and labeled nucleotides, provided the unit specific marker and nucleotides are differentially labeled with the at least two distinguishable detectable labels that are a FRET fluorophore pair, and

analyzing the single nucleic acid molecule for a coincident event using a single molecule detection system,

wherein the coincidence event indicates that the at least two distinguishable detectable labels are bound to the single nucleic acid molecule.

24. The method of claim 23, wherein one detectable label is attached to the unit specific marker and is a first FRET fluorophore, and the other detectable label is incorporated into a new nucleic acid molecule hybridized to the single nucleic acid molecule and is the donor or acceptor of the first FRET fluorophore.