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Publication Number
US-9045798-B1
Publication Date
2015-06-02
Expiration Date
Abstract
The present invention provides novel compositions, methods and apparatus for DNA sequencing that can be performed, e.g., in a two-electrode chamber. The present invention also provides a method for sequencing a nucleic acid comprising immobilizing a plurality of complexes comprising a target nucleic acid, a primer nucleic acid, and a polymerase onto a surface, contacting the surface with a plurality of charged particles comprising a nucleotide phosphate by applying an electric field, reversing the electric field to transport unbound charged particles away from the surface, and detecting the incorporation of a nucleotide phosphate into a single molecule of the primer nucleic acid.
Core Innovation
The invention relates to increasing the signal to noise ratio (S/N) for single particle detection while sequencing a nucleic acid. A plurality of complexes comprising a target nucleic acid, a primer nucleic acid, and a polymerase are immobilized onto a surface, and a sequencing reaction mixture including single particles is provided for the sequencing.
The sequencing reaction mixture includes single particles that are dendrimers. Each dendrimer particle comprises both a plurality of dyes and a plurality of incorporatable substrate nucleotide phosphates for the polymerase, and the dendrimer particles sequentially reacts with an immobilized complex.
In each sequential reaction, one nucleotide phosphate reacts with the polymerase, is cleaved from the dendrimer, and is incorporated into the primer nucleic acid. By releasing the dendrimer from the complex, the method supports detecting the single particle while incorporating each nucleotide phosphate, with dye-loaded dendrimer single particles and detection modalities to support low-background single-molecule detection during nucleotide incorporation.
Claims Coverage
The document provides one independent claim directed to a method for increasing signal to noise ratio during single particle nucleic-acid sequencing. The main inventive features are expressed through immobilized target/primer/polymerase complexes, dendrimer single particles carrying multiple dyes and incorporatable nucleotide phosphate substrates, and sequencing-coupled detection during incorporation with dendrimer release.
Signal-to-noise improved single particle sequencing with immobilized complexes
A method for increasing S/N for single particle detection while sequencing a nucleic acid by immobilizing a plurality of complexes comprising a target nucleic acid, a primer nucleic acid, and a polymerase onto a surface.
Dendrimer single particles with dyes and incorporatable nucleotide phosphates
Providing a sequencing reaction mixture including dendrimer single particles, each dendrimer comprising a plurality of dyes and a plurality of incorporatable substrate nucleotide phosphates for the polymerase, where dendrimer particles sequentially reacts with an immobilized complex.
Sequential nucleotide phosphate reaction, cleavage, and primer incorporation with dendrimer release
In each sequential reaction, one of the plurality of nucleotide phosphates reacts with the polymerase, is cleaved from the dendrimer, and is incorporated into the primer nucleic acid, thereby releasing the dendrimer from the complex.
Detecting the single particle during incorporation of nucleotide phosphates
Detecting the single particle while incorporating each nucleotide phosphate thereby sequencing the target nucleic acid.
Across the independent claim, the core inventive coverage centers on immobilized target/primer/polymerase complexes, dendrimer single particles carrying multiple dyes and incorporatable nucleotide phosphates, sequential cleavage/incorporation that releases the dendrimer, and detection coupled to nucleotide incorporation to improve S/N.
Stated Advantages
Increasing the signal to noise ratio (S/N) for single particle detection while sequencing a nucleic acid.
Documented Applications
Single particle detection while sequencing a nucleic acid using dendrimer single particles and immobilized complexes.
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