System and methods for nucleic acid sequencing of single molecules by polymerase synthesis

摘要

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.

主权项

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.