초록

This invention relates to improved methods for sequencing and genotyping nucleic acid in a single molecule configuration. The method involves single molecule detection of fluorescent labeled PPi moieties released from NTPs as a polymerase extension product is created.

대표청구항 ▼

1. A method for sequencing a target nucleic acid molecule in a system having a flow chamber with a support, said method comprising: (a) providing a flow chamber with a support comprising an array of immobilized complexes, each of said immobilized complexes comprising a nucleic acid polymerizing enzy

1. A method for sequencing a target nucleic acid molecule in a system having a flow chamber with a support, said method comprising: (a) providing a flow chamber with a support comprising an array of immobilized complexes, each of said immobilized complexes comprising a nucleic acid polymerizing enzyme, a target nucleic acid, and a primer which complements a region of the target nucleic acid, wherein the nucleic acid polymerizing enzyme is complexed with the target nucleic acid and the primer, and either the nucleic acid polymerizing enzyme or the target nucleic acid is immobilized on the support;(b) introducing into the chamber a plurality of nucleotide analogs with labels on their γ-phosphates that are not positioned on said support to move through said flow chamber to contact said complexes;(c) extending the primer by incorporating nucleotide analogs from the plurality of nucleotide analogs into the primer, thereby forming a polymerase extension product, which is a complement of the target nucleic acid; and(d) sequencing the target nucleic acid by individually detecting the labels of the γ-phosphates from the nucleotide analogs during a process in which the polymerase extension product is formed. 2. The method of claim 1, wherein said labels are attached to the γ-phosphates via a linker. 3. The method of claim 1, wherein said labels are directly attached to the γ-phosphates. 4. The method of claim 1, wherein said flow chamber is made of plastic or glass. 5. The method of claim 1, wherein said individually detecting the labels of the γ-phosphates from the nucleotide analogs is performed using a detector which is capable of detecting a single molecule. 6. The method of claim 5, wherein said detector is 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 coupled device, a near-field scanning microscope, a far-field confocal microscope, a microscope that detects wide-field epi-illumination, and a total internal reflection fluorescence microscope when the labels are fluorescent labels. 7. The method of claim 1, wherein the nucleic acid polymerizing enzyme or the target nucleic acid is present as an individual molecule located in a defined location of the support. 8. The method of claim 1, wherein each of the immobilized complexes of said array is different. 9. The method of claim 1, wherein said array of immobilized complexes is addressable. 10. The method of claim 5, wherein said detector is capable of acquiring a sequence of images. 11. The method of claim 10, wherein said sequence of images is analyzed to track single molecules. 12. The method of claim 5, wherein said detector provides low background and enables the detection of single molecules. 13. The method of claim 5, wherein said detector detects the labels in an evanescent optical field when the labels are fluorescent labels. 14. The method of claim 5, wherein said detector detects the labels via 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 when the labels are fluorescent labels.