Composition and method for nucleic acid sequencing
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.
1. A method for sequencing a single target nucleic acid molecule, said method comprising:
i. immobilizing onto a support a polymerase nucleic acid complex comprising the single target nucleic acid molecule, a primer nucleic acid which complements a region of the single target nucleic acid molecule, at least one family A or family B nucleic acid polymerase, and an attachment complex, wherein said attachment complex:
(a) is associated with the support;
(b) attaches to said polymerase on both sides of a DNA binding cleft of the polymerase; and
(c) entraps the single target nucleic acid molecule, to produce an immobilized polymerase nucleic acid complex;
ii. contacting said immobilized polymerase nucleic acid complex with at least one type of labeled nucleotide triphosphate (NTP), wherein each NTP is labeled with a detectable label;
iii. sequentially detecting the incorporation of a plurality of said at least one type of labeled NTP into a single molecule of said primer nucleic acid to generate a nucleotide sequence; and
iv. determining the complement to the nucleotide sequence generated in step iii, wherein the complement is a nucleotide sequence of the single target nucleic acid molecule, thereby sequencing the single target nucleic acid molecule.
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 3 , 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 3 , 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 1 , 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 1 , wherein the single target nucleic acid molecule is a circular nucleic acid molecule.
13. The method of claim 12 , wherein the attachment complex irreversibly associates the circular nucleic acid molecule with the polymerase.