Pyrophosphorolytic sequencing

Abstract

A method for determining the sequence of a target nucleic acid, including steps of contacting a target nucleic acid with a polymerase to sequentially remove nucleotide triphosphates from the target nucleic acid, wherein the nucleotide triphosphates that are removed have a variety of different base moieties; and distinguishing the different base moieties for the nucleotide triphosphates that are removed. Also provided is a apparatus including a nanopore positioned in a fluid impermeable barrier to form a passage through which a nucleotide triphosphate can pass from a first fluid reservoir to a second fluid reservoir, and a reaction mix in the first fluid reservoir that includes a polymerase, target nucleic acid having two strands, and pyrophosphorolytic concentration of pyrophosphate.

Description

[0001]This application is based on, and claims the benefit of, U.S. Provisional Application No. 61 / 827,175, filed May 24, 2013, which is incorporated herein by reference.BACKGROUND[0002]This disclosure relates generally to nucleic acid analysis, and more specifically to nucleic acid synthesis using nanopores.[0003]Currently available commercial platforms for sequencing DNA are relatively costly. These platforms use a ‘sequencing by synthesis’ approach, so called because DNA polymers are synthesized while detecting the addition of each monomer (i.e. nucleotide) to the growing polymer structure. Because a template DNA strand strictly directs synthesis of a new DNA polymer, one can infer the sequence of the template DNA from the series of nucleotide monomers that were added to the growing strand during the synthesis. The ability to detect monomer additions is facilitated by specially engineered variants of the biochemical components that normally carry out DNA synthesis in biological s...

AI Technical Summary

Benefits of technology

The present patent provides a method and apparatus for determining the sequence of a target nucleic acid by contacting it with a polymerase to remove nucleotide triphosphates with different base moieties and detecting them. This technology can speed up sequencing with greater accuracy and efficiency, and has potential applications in areas such as genomic research and medical diagnostics.

Problems solved by technology

Currently available commercial platforms for sequencing DNA are relatively costly.

These engineered components are expensive to make and are consumed in relatively large amounts during sequencing by synthesis.

Furthermore, monitoring the reaction uses relatively expensive hardware such as lasers, detection optics and complex fluid delivery systems.

The most successful commercial platforms to date also require expensive reagents and hardware to amplify the DNA templates before sequencing by synthesis can even begin.

However, the resulting variations in the ionic current flowing through the nanopores are quite small and it is difficult to distinguish one nucleotide from another.

However despite these efforts, nanopore-exonuclease sequencing has not yet been demonstrated at a commercially viable level to date.

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