Pyrophosphorolytic sequencing
AssigneeIllumina Cambridge Limited
InventorWouter MEULEMAN
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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.
Claims24
A method for determining the sequence of a target nucleic acid, comprising
- (a) providing a target nucleic acid having two strands;
- (b) contacting the target nucleic acid with a polymerase under conditions to sequentially remove nucleotides from the first of the two strands by pyrophosphorolysis, thereby sequentially producing nucleotide triphosphates having a variety of different base moieties;
- and
- (c) distinguishing the different base moieties for the sequentially produced nucleotide triphosphates, thereby determining the sequence of the target nucleic acid.
A apparatus, comprising
- (a) a fluid impermeable barrier separating a first fluid reservoir from a second fluid reservoir;
- (b) a nanopore positioned in the fluid impermeable barrier to form a passage through which a nucleotide triphosphate can pass from the first fluid reservoir to the second fluid reservoir;
- and
- (c) a reaction mix in the first fluid reservoir, the reaction mix comprising a polymerase, target nucleic acid having two strands, and pyrophosphorolytic concentration of pyrophosphate.
A method for determining the sequence of a target nucleic acid, comprising
- (a) providing a target nucleic acid;
- (b) contacting the 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
- (c) distinguishing the different base moieties for the nucleotide triphosphates that are removed, thereby determining the sequence of the target nucleic acid.
Dependencies are inferred from the claim text.