A method of incorporating a nucleotide with a base complementary to the next base in a template strand immediately downstream of a primer in a primed template nucleic acid molecule, the method comprising the steps of:
- (a) contacting the primed template nucleic acid molecule with a first reaction mixture that comprises a DNA polymerase, thereby forming a binary complex comprising the primed template nucleic acid molecule and the DNA polymerase;
- (b) contacting the binary complex from step (a) with a second reaction mixture that comprises the DNA polymerase and a first test nucleotide, without incorporating the first test nucleotide into the primer, thereby forming a second complex comprising the primed template nucleic acid molecule and the DNA polymerase;
- (c) measuring binding of the primed template nucleic acid molecule to the DNA polymerase at one or more points during each of steps (a) and (b), without chemical incorporation of the first test nucleotide into the primer, to establish whether the second complex that formed in step (b) is a ternary complex comprising the first test nucleotide;
- (d) selecting one of the following two options,
- (i) if it is established in step (c) that the second complex is not the ternary complex, then contacting the second complex from step (b) with a third reaction mixture that comprises the DNA polymerase in combination with the first test nucleotide and a second test nucleotide, the second test nucleotide being different from the first test nucleotide;
- and
- (ii) if it is established in step (c) that the second complex is the ternary complex, then performing an incorporation reaction to incorporate the first test nucleotide into the primer without first contacting the primed template nucleic acid molecule from step (b) with the third reaction mixture,
- wherein either
- the first reaction mixture is free of the first test nucleotide, or
- the first and second reaction mixtures each comprise the first test nucleotide, the first test nucleotide of the first reaction mixture being present at a concentration lower than the concentration of the first test nucleotide of the second reaction mixture.