NUCLEIC ACID SEQUENCING METHODS AND SYSTEMS

摘要

The present disclosure provides compositions, methods and systems for sequencing a template nucleic acid using a polymerase based, nucleic acid binding reaction involving examination of the interaction between a polymerase and template nucleic acid in the presence of one or more unlabeled nucleotides. The methods rely, in part, on identifying a base of a template nucleic acid during nucleic acid synthesis by controlling the sequencing reaction conditions. Template nucleic acid bases may be identified during an examination step followed by an optional incorporation step.

主权项

1. A method for sequencing a template nucleic acid molecule, the method comprising:
an examination step comprising: (a) providing a template nucleic acid molecule primed with a primer; (b) contacting the primed template nucleic acid molecule with a first reaction mixture comprising a polymerase and a first unlabeled nucleotide molecule, wherein the primed template nucleic acid molecule, the polymerase and the first unlabeled nucleotide molecule are capable of forming a ternary complex when the first unlabeled nucleotide molecule is complementary to a next base of the primed template nucleic acid molecule, and wherein the primed template nucleic acid molecule and the polymerase are capable of forming a binary complex when the first unlabeled nucleotide molecule is not complementary to a next base of the primed template nucleic acid molecule, (c) monitoring the interaction of the polymerase with the primed template nucleic acid molecule in the presence of the first unlabeled nucleotide molecule, and without chemical incorporation of the first unlabeled nucleotide molecule into the primer of the primed template nucleic acid molecule; and (d) identifying the nucleotide that is complementary to the next base of the primed template nucleic acid molecule by the monitored interaction of step (c).