COMPOSITION AND METHOD FOR NUCLEIC ACID SEQUENCING

• US 20090068655A1
• Filed: 10/23/2007
• Published: 03/12/2009
• Est. Priority Date: 04/08/2003
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience Labs (L'Oreal SA)
• Inventors: Williams, John G.K.
• Granted Patent: US 7,858,311 B2
• 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 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2523/101 Crosslinking agents, e.g. psoralen
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The present invention provides compositions and methods for detecting incorporation of a labeled nucleotide triphosphate onto the growing end of a primer nucleic acid molecule. The method is used, for example, to genotype and sequence a nucleic acid. In a preferred embodiment, the method described herein detects individual NTP molecules.

Claims

1. A polymerase-nucleic acid complex, said polymerase-nucleic acid complex comprising:

a target nucleic acid and a nucleic acid polymerase, wherein said polymerase has an attachment complex comprising at least one anchor, which said at least one anchor irreversibly associates said target nucleic acid with said polymerase to increase the processivity index.

2. The polymerase-nucleic complex of claim 1, wherein said polymerase-nucleic acid complex further comprises a primer nucleic acid which complements a region of said target nucleic acid.

3. The polymerase-nucleic complex of claim 1, wherein said attachment complex comprises at least two anchors.

4. The polymerase-nucleic complex of claim 3, wherein said attachment complex is attached to a support.

5. The polymerase-nucleic complex of claim 1, wherein said attachment complex comprises a topological tether.

6. The polymerase-nucleic complex of claim 3, wherein said at least two anchors further comprises a topological tether.

7. The polymerase-nucleic complex of claim 6, wherein said topological tether is attached to at least one anchor via a complementary binding pair.

8. The polymerase-nucleic complex of claim 6, wherein said topological tether is attached to at least two anchors via at least two complementary binding pairs.

9. The polymerase-nucleic complex of claim 7, wherein said complementary binding pairs are selected from the group consisting of any haptenic or antigenic compound in combination with a corresponding antibody or binding portion or fragment thereof, nonimmunological binding pairs, receptor-receptor agonist or antagonist, IgG-protein A, lectin-carbohydrate, enzyme-enzyme cofactor, enzyme-enzyme-inhibitor, and complementary polynucleotide pairs capable of forming nucleic acid duplexes.

10. The polymerase-nucleic complex of claim 9, wherein said complementary binding pair is selected from the group consisting of digoxigenin and anti-digoxigenin, fluorescein and anti-fluorescein, dinitrophenol and anti-dinitrophenol, bromodeoxyuridine and anti-bromodeoxyuridine, mouse immunoglobulin and goat anti-mouse immunoglobulin, biotin-avidin, biotin-streptavidin, thyroxine and cortisol, a phenylalanine derivative and hydrazine linker and acetylcholine and receptor-acetylcholine.

11-28. -28. (canceled)

29. A method for detecting incorporation of at least one NTP into a single primer nucleic acid molecule, said method comprising:

i. immobilizing onto a support a polymerase nucleic acid complex comprising a target nucleic acid, a primer nucleic acid which complements a region of the target nucleic acid, at least one nucleic acid polymerase, wherein said nucleic acid polymerase has at least one anchor;

ii. contacting said immobilized complex with at least one type of labeled nucleotide triphosphate [NTP], wherein each NTP is labeled with a detectable label, and

iii. detecting the incorporation of said at least one type of labeled NTP into a single molecule of said primer, while said at least one type of labeled NTP is in contact with said immobilized complex, by detecting the label of the NTP while said at least one type of labeled NTP is in contact with said polymerase nucleic acid complex.

30. The method of claim 29, wherein said polymerase nucleic acid complex is contacted with a single type of labeled NTP.

31. The method of claim 29, wherein said polymerase nucleic acid complex is contacted with at least two different types of NTPs, and wherein each type of NTP is uniquely labeled.

32. The method of claim 29, wherein said polymerase nucleic acid complex is contacted with at least four different types of NTPs, and wherein each type of NTP is uniquely labeled.

33. The method of claim 29, wherein said NTPs are labeled on the γ

-phosphate.

34. The method of claim 33, wherein said NTPs are labeled on the γ

-phosphate with a fluorescent label.

35. The method of claim 29, wherein the detecting comprises detecting a unique signal from the labeled NTP using a system or device selected from the group consisting of an optical reader, a high-efficiency photon detection system, a photodiode, a camera, a charge couple device, an intensified charge couple device, a near-field scanning microscope, a far-field confocal microscope, a microscope that detects wide-field epi-illumination, evanescent wave excitation and a total internal reflection fluorescence microscope.

36. The method of claim 29, wherein the label of the NTP is detected using a method comprising a four color evanescent wave excitation device.

37. The method of claim 29, wherein said detecting is carried out by a mechanism selected from the group consisting of fluorescence resonance energy transfer, an electron transfer mechanism, an excited-state lifetime mechanism and a ground-state complex quenching mechanism.

38. The method of claim 29, wherein said detecting step comprises measuring a residence time of a labeled NTP in said polymerase nucleic acid complex.

39-46. -46. (canceled)