Composition and method for nucleic acid sequencing

• US 7,939,256 B2
• Filed: 10/23/2007
• Issued: 05/10/2011
• Est. Priority Date: 04/08/2003
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Williams, John G. K.
• Primary Examiner(s): Strzelecka; Teresa E
• Application Number:
  US11/877,628
• Publication Number:
  US 20090092970A1
• Time in Patent Office: 1,295 Days
• Field of Search: None
• US Class Current: 435/6.12
• 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 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, and at least one family A or family B polymerase, wherein said polymerase has an attachment complex that attaches to said polymerase on both sides of a DNA binding cleft and that increases processivity of said polymerase;

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.

2. The method of claim 1, wherein said polymerase nucleic acid complex is contacted with a single type of labeled NTP.

3. The method of claim 1, 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.

4. The method of claim 1, 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.

5. The method of claim 1, wherein said NTPs are labeled on the γ

-phosphate.

6. The method of claim 5, wherein said NTPs are labeled on the γ

-phosphate with a fluorescent label.

7. The method of claim 1, 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.

8. The method of claim 1, wherein the label of the NTP is detected using a method comprising a four color evanescent wave excitation device.

9. The method of claim 1, 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.

10. The method of claim 1, wherein said detecting step comprises measuring a residence time of a labeled NTP in said polymerase nucleic acid complex.

11. The method of claim 10, wherein the residence time of an NTP that is incorporated into the primer nucleic acid is longer than the residence time of an incorrectly paired NTP that is not incorporated.

12. The method of claim 11, wherein the residence time of an NTP that is incorporated into the primer nucleic acid is at least 200 times longer to about 500 times longer than the residence time of an incorrectly paired NTP that is not incorporated.

13. The method of claim 10, wherein the residence time of an NTP that is incorporated into the primer nucleic acid is about 1.0 milliseconds to about 100 milliseconds.

14. The method of claim 13, wherein the residence time of an NTP that is incorporated into the primer nucleic acid is about 2.0 milliseconds to about 10 milliseconds.

15. The method of claim 1, further comprising the step of

genotyping said target nucleic acid by determining the identity of at least one NTP that is incorporated into a single molecule of the primer.

16. The method of claim 1, further comprising:

sequencing said target nucleic acid by determining the identity and sequence of incorporation of NTPs that are incorporated into a single molecule of the primer.

17. The method of claim 1, wherein said detection is a sequential detection of the identities of more than one uniquely labeled dNTPs that are sequentially incorporated into the primer, wherein said sequential detection yields the sequence of region of the target DNA that is downstream of the elongating end of the primer.