Apparatus and methods for kinetic analysis and determination of nucleic acid sequences

摘要

A method of distinguishing nucleotide sequences for different nucleic acid molecules including the steps of (a) mixing a plurality of different nucleic acid molecules with polymerase molecules and nucleotide molecules, wherein the different nucleic acid molecules are attached to a surface in the form of an array of nucleic acid features; (b) determining a transient state of the polymerase molecules at the nucleic acid features; and (c) identifying a subset of nucleic acid features that correctly incorporate the nucleotide molecules based on the transient state of the polymerase molecules at the nucleic acid features, thereby distinguishing the nucleotide sequences for the different nucleic acid molecules.

主权项

1. A method of distinguishing nucleotide sequences for different nucleic acid molecules, comprising
(a) mixing a plurality of different nucleic acid molecules with polymerase molecules and nucleotide molecules to cause binding of the polymerase molecules to the nucleic acid molecules, wherein the different nucleic acid molecules are attached to a surface in the form of an array of nucleic acid features at a density of at least 100 features/cm2, and wherein the mixing comprises stopped flow delivery of the polymerase molecules to the nucleic acid features; (b) monitoring the binding of the polymerase molecules to the nucleic acid features at several points during a pre-equilibrium time period, thereby determining a transient state of the polymerase molecules at the nucleic acid features; and (c) identifying nucleic acid features of the array that correctly incorporate the nucleotide molecules based on the transient state of the polymerase molecules at the nucleic acid features, thereby distinguishing the nucleotide sequences for the different nucleic acid molecules.