DNA sequencing by synthesis using modified nucleotides and nanopore detection

摘要

Method for determining the nucleotide sequence of a single stranded nucleic acid molecule comprising: (a) contacting the single stranded nucleic acid molecule, in which the single stranded nucleic acid molecule is in an electrolyte solution in contact with a nanopore in a membrane and in which the single stranded nucleic acid molecule has a primer hybridized with a part thereof, with a nucleic acid polymerase and at least four nucleotide polyphosphate analogues (NPP) labeled with phosphate under conditions that allow that the nucleic acid polymerase catalyzes the incorporation of one of the NPP analogs into the primer if it is complementary to the nucleotide residue of the single stranded nucleic acid molecule that is immediately 5 'from a nucleotide residue of the acid molecule single stranded nucleic acid hybridized with the 3'-terminal nucleotide residue of the primer, to form a nucleic acid extension product and with the proviso that (i) the type of base in each NPP analog is different from the type of base in each of the other three NPP analogs, and (ii) or the number of phosphates in the polyphosphate part of each NPP analog is different from the number of phosphates in the polyphosphate part of each of the other three NPP analogs, or the number of phosphates in the polyphosphate part of each of the Four NPP analogs are the same and the type of tag on each NPP analog is different from the type of tag on each of the other three NPP analogs, in which the incorporation of the NPP analog results in the release of a polyphosphate having the label attached to it; and (b) determine which NPP analog has been incorporated into the primer to form a nucleic acid extension product in step (a) by applying a voltage across the membrane and measuring an electronic change through the nanopore resulting from that the polyphosphate having the label attached to it generated in step (a) is translocated through the nanopore, in which the electronic change is different for each number of phosphates in the polyphosphate part, or for each type of different label , as appropriate, thereby identifying the nucleotide residue in the single stranded nucleic acid complementary to the incorporated NPP analog; and (c) iteratively perform steps (a) and (b) for each nucleotide residue of the single stranded nucleic acid molecule that is being sequenced, in which the analog of each stage (a) is incorporated NPP in the nucleic acid extension product resulting from the previous iteration of step (a) if it is complementary to the nucleotide residue of the single stranded nucleic acid molecule that is immediately 5 'from a nucleotide residue of the molecule of single stranded nucleic acid hybridized with the 3'-terminal nucleotide residue of the nucleic acid extension product, thereby determining the nucleotide sequence of the single stranded nucleic acid molecule.