High accuracy nanopore-based single molecule sequencing by synthesis with tagged nucleotides

A method for sequencing a nucleic acid molecule, the method comprising:

• (a) providing a chip comprising a plurality of individually addressable nanopores, wherein an individually addressable nanopore of said plurality of individually addressable nanopores comprises a nanopore in a membrane that is disposed adjacent to an electrode, wherein said nanopore is linked to a nucleic acid polymerase, and wherein each individually addressable nanopore is adapted to select a tag attached to a tagged unincorporable nucleotide from a set of four possible separately tagged unincorporable nucleotides;
• (b) directing said nucleic acid molecule adjacent to or in proximity to said nanopore along with a nucleic acid primer complementary to a portion of said nucleic acid template molecule;
• (c) providing four types of unincorporable tagged nucleotides (A, C, G, T) and four types of untagged nucleotide reversible terminators (A, C, G, T) into a reaction chamber comprising each individual said nanopore;
• wherein each type of the unincorporable tagged nucleotides comprises a different tag, such that each tag, when captured in said nanopore, elicits a distinct ionic current blockade signal;
• (d) carrying out a polymerization reaction with the aid of a polymerase;
• (e) detecting the tag on any unincorporable tagged nucleotide that binds to the nucleic acid molecule with the aid of said electrode, which event will occur multiple times, when such unincorporable tagged nucleotides bind and when such tag is captured within the channel of said nanopore, until an untagged nucleotide reversible terminator is bound and is polymerized to the primer strand along said nucleic acid molecule to generate a strand that is complementary to at least a portion of said nucleic acid molecule;
• wherein said nucleotide reversible terminators have a blocking group at the 3′ hydroxyl position;
• (f) cleaving the 3′ blocking group on said polymerized nucleotide reversible terminator in such a way as to regenerate the 3′ hydroxyl group on said nucleotide reversible terminator;
• (g) repeating (c) to (f) as long as the polymerization along the said nucleic acid molecule continues,
• thereby obtaining the sequence of said nucleic acid molecule.

A method for sequencing a nucleic acid molecule, the method comprising:

• (a) providing a chip comprising a plurality of individually addressable nanopores, wherein an individually addressable nanopore of said plurality of individually addressable nanopores comprises a nanopore in a membrane that is disposed adjacent to an electrode, wherein said nanopore is linked to a nucleic acid polymerase, and wherein each individually addressable nanopore is adapted to select a tag attached to a tagged nucleotide reversible terminator from a set of four possible separately tagged nucleotide reversible terminators (A, C, G and T) bearing tags 1, 2, 3 and 4 respectively;
• wherein said tagged nucleotide reversible terminators have a blocking group at the 3′ hydroxyl position, and said tag at the terminal phosphate position;
• (b) directing said nucleic acid molecule adjacent to or in proximity to said nanopore along with a nucleic acid primer complementary to a portion of said nucleic acid template molecule;
• (c) providing four types of tagged nucleotide reversible terminators (A, C, G, T) into a reaction chamber comprising each individual said nanopore;
• wherein said nucleotide reversible terminators have a blocking group at the 3′ hydroxyl position;
• (d) carrying out a polymerization reaction with the aid of a polymerase;
• (e) detecting the tag on any unincorporable tagged nucleotide that binds to the nucleic acid molecule with the aid of said electrode, when such unincorporable tagged nucleotides bind and when said tag is captured within the channel of said nanopore, which event will reveal the specific nucleotide reversible terminator polymerized along said nucleic acid molecule;
• (f) cleaving the 3′ blocking group on said polymerized nucleotide reversible terminator in such a way as to regenerate the 3′ hydroxyl group on said nucleotide reversible terminator;
• (g) repeating (c) to (f) as long as the polymerization along the said nucleic acid molecule continues,
• thereby obtaining the sequence of said nucleic acid molecule.