US 12,467,085 B2
High accuracy nanopore-based single molecule sequencing by synthesis with tagged nucleotides
Jingyue Ju, Englewood Cliffs, NJ (US); Carl W. Fuller, Berkeley Heights, NJ (US); Shiv Kumar, Belle Mead, NJ (US); Sergey Kalachikov, Bronx, NY (US); Xiaoxu Li, New York, NY (US); Irina Morozova, Bronx, NY (US); James Russo, New York, NY (US); and Steffen Jockusch, New York, NY (US)
Assigned to THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF NEW YORK, New York, NY (US)
Appl. No. 17/436,513
Filed by Jingyue Ju, Englewood Cliffs, NJ (US); Carl W. Fuller, Berkeley Heights, NJ (US); Shiv Kumar, Belle Mead, NJ (US); Sergey Kalachikov, Bronx, NY (US); Xiaoxu Li, New York, NY (US); Irina Morozova, Bronx, NY (US); James Russo, New York, NY (US); and Steffen Jockusch, New York, NY (US)
PCT Filed Mar. 5, 2020, PCT No. PCT/US2020/021253
§ 371(c)(1), (2) Date Sep. 3, 2021,
PCT Pub. No. WO2020/181129, PCT Pub. Date Sep. 10, 2020.
Claims priority of provisional application 62/815,250, filed on Mar. 7, 2019.
Prior Publication US 2022/0177959 A1, Jun. 9, 2022
Int. Cl. C12Q 1/68 (2018.01); C12Q 1/6869 (2018.01)
CPC C12Q 1/6869 (2013.01) 20 Claims
 
1. A method for sequencing a nucleic acid, wherein the method comprises:
(a) providing a chip comprising a plurality of individually addressable nanopores, wherein the 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;
(b) directing said nucleic acid adjacent to or in proximity to said nanopore along with a nucleic acid primer complementary to a portion of said nucleic acid;
(c) providing four types of unincorporable tagged nucleotides (A, C, G, T) and four types of incorporable tagged nucleotides (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, and wherein each incorporable tagged nucleotide comprises a single fifth tag, which is distinct from the four tags on the unincorporable tagged nucleotides, and which produces a fifth unique ionic current blockade signal;
(d) carrying out a polymerization reaction with the aid of the nucleic acid polymerase;
(e) detecting the tag on any unincorporable tagged nucleotide that binds to the nucleic acid with the aid of said electrode, which event will occur multiple times consecutively, when such unincorporable tagged nucleotides bind and when such tag is captured within the channel of said nanopore;
(f) detecting the tag on any incorporable tagged nucleotide that binds to the nucleic acid with the aid of said electrode, which event will occur one time, wherein the tag is detected prior to its being released from said incorporable tagged nucleotide as the incorporable tagged nucleotide is polymerized along said nucleic acid to generate a strand that is complementary to at least a portion of said nucleic acid, when such tag is captured within the channel of said nanopore;
(g) repeating (e) and (f) as long as the polymerization along the said nucleic acid continues,
thereby obtaining the sequence of said nucleic acid.