Polymerase-template complexes
AssigneeRoche Sequencing Solutions, Inc.
InventorsAruna Ayer, Preethi Sarvabhowman, Charles Schwab
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Abstract
The present disclosure provides methods and compositions for enhancing the processivity of a polymerase in catalyzing template-dependent DNA synthesis in high concentrations of salt. Also disclosed are methods and compositions for enhancing the assembly of polymerase-template complex compatible with active DNA synthesis in the presence of low levels of nucleotides and at a high temperature, such as temperatures at or near the melting temperature of the polymerase.
Claims20
A method for preparing a polymerase-template complex, the method comprising incubating a primed DNA template with a molar excess of a DNA polymerase in a solution having up to 2.2 μM nucleotide polyphosphate for a period of time sufficient to allow the DNA polymerase and the DNA template to form the polymerase-template complex, wherein the incubation is performed at or above the melting temperature of the free polymerase and below the melting temperature of the DNA polymerase complexed with the DNA template.
A method of sequencing a DNA template, the method comprising:
- (a) forming a primed DNA template by annealing a primer to the DNA template;
- (b) forming a polymerase-template complex by incubating a primed DNA template with a molar excess of a DNA polymerase in a solution having up to 2.2 μM nucleotide polyphosphate for a period of time sufficient to allow the DNA polymerase and the DNA template to form the polymerase-template complex, wherein the incubation is performed at or above the melting temperature of the free polymerase and below the melting temperature of the DNA polymerase complexed with the DNA template;
- (c) forming a nanopore-sequencing complex comprising:
- (c1) a nanopore deposited in a membrane, wherein the nanopore is positioned adjacent to or in proximity to a sensing electrode of a biochip;
- and
- (c2) the polymerase-template complex, wherein the DNA polymerase of the polymerase-template complex is linked to the nanopore;
- (d) providing tagged nucleotides to the nanopore sequencing complex to initiate a primer extension reaction that polymerizes the tagged nucleotides into a nucleic acid complementary to the DNA template (“complementary strand”);
- and
- (e) detecting tags associated with tagged nucleotides incorporated into the complementary strand by measuring a current flowing through the nanopore;
- and
- (f) deriving the sequence of the DNA template from the measured current flowing through the nanopore.
Dependencies are inferred from the claim text.