US 12,188,086 B2
Nucleotide analogues and use thereof for nucleic acid sequencing and analysis preliminary class
Jingyue Ju, Englewood Cliffs, NJ (US); Xiaoxu Li, New York, NY (US); Shiv Kumar, Belle Mead, NJ (US); Xin Chen, Hunan (CN); James J. Russo, New York, NY (US); Minchen Chien, Tenafly, NJ (US); Steffen Jockusch, New York, NY (US); Chuanjuan Tao, Fort Lee, NJ (US); Xuanting Wang, Braintree, MA (US); Sergey Kalachikov, Bronx, NY (US); Irina Morozova, Bronx, NY (US); and Shundi Shi, Ozone Park, NY (US)
Assigned to THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF NEW YORK, New York, NY (US)
Appl. No. 16/980,708
Filed by The Trustees of Columbia University in the City of New York, New York, NY (US)
PCT Filed Mar. 14, 2019, PCT No. PCT/US2019/022326
§ 371(c)(1), (2) Date Sep. 14, 2020,
PCT Pub. No. WO2019/178393, PCT Pub. Date Sep. 19, 2019.
Claims priority of provisional application 62/643,633, filed on Mar. 15, 2018.
Prior Publication US 2021/0139976 A1, May 13, 2021
Int. Cl. C12Q 1/68 (2018.01); C07H 19/00 (2006.01); C07H 21/04 (2006.01); C12Q 1/6874 (2018.01); C12Q 1/6876 (2018.01)
CPC C12Q 1/6874 (2013.01) [C07H 19/00 (2013.01); C07H 21/04 (2013.01); C12Q 1/6876 (2013.01); C12Q 2563/107 (2013.01)] 21 Claims
 
1. A method of sequencing nucleic acid comprising:
a) providing at least one nucleic acid template hybridized to a primer;
b) extending the primer hybridized to said nucleic acid template with a polymerase, and a set of nucleotide analogues selected from the group consisting of:
i) a set of fluorescently labeled nucleotide analogues, wherein said fluorescently labeled nucleotide analogues have a fluorescent label linked to the base via a cleavable linker and a blocking group on the 3′-hydroxyl position, wherein different fluorescently labeled nucleotide analogues in the set may have different fluorescent labels and different cleavable linkers;
ii) a set of anchor labeled nucleotide analogues, wherein said anchor labeled nucleotide analogues have an anchor attached to the base via a cleavable linker and a blocking group on the 3′-hydroxyl position, wherein different anchor labeled nucleotide analogues in the set may have different anchors and different cleavable linkers; and
iii) a set comprising a combination of both fluorescently and anchor labeled nucleotide analogues, wherein said fluorescently or anchor labeled nucleotide analogues have the fluorescent or anchor label linked to the base via a cleavable linker and a blocking group on the 3′-hydroxyl position, wherein different nucleotide analogues in the set may have different fluorescent labels, anchors, and/or cleavable linkers;
c) extending any unextended primer with another set of nucleotide analogues having a blocking group on the 3′-hydroxyl position without any base modifications, and if the set of nucleotide analogues in step b) has one or more fluorescently labeled nucleotide analogues, identifying one or more fluorescence signals due to incorporation of one or more fluorescently labeled nucleotide analogues;
d) if the set of nucleotide analogues in step b) comprises anchor labeled nucleotide analogues:
(i) labeling the anchor labeled nucleotide analogues with one or more corresponding fluorescently or quantum dot labeled anchor binding molecules;
(ii) identifying newly generated fluorescence signals to thereby partially or completely identify the incorporated nucleotide analogues due to the labeling carried out in step d) (i); and
(iii) if not all of the anchor labeled nucleotide analogues in step a) are labeled in step d) (i), repeating steps d) (i) and d) (ii);
e) cleaving one or more of the fluorescent labels from one or more of the fluorescently labeled nucleotide analogues in step b) or one or more of the fluorescently or quantum dot labeled anchor binding molecules in step d) (i) with a specific cleavable agent that cleaves one type of the cleavable linkers, wherein the specific cleavable agent does not cleave the blocking group on the 3′-hydroxyl position of the nucleotide analogues;
f) identifying loss of fluorescence signals due to the cleavage carried out in step e) to partially or completely identify the incorporated nucleotide analogues;
g) if not all of the fluorescent labels from fluorescently labeled nucleotide analogues in step b) or the fluorescently or quantum dot labeled anchor binding molecules from step d) (i) are cleaved, repeating steps e) and f) with a different cleavable agent;
h) determining the specific nucleotide analogue incorporated by comparing the results obtained in steps c, d) (ii) and f);
i) cleaving any remaining fluorescent labels or anchors from the extended primer, at the same time cleaving the blocking group on the 3′-hydroxyl position of the incorporated nucleotide analogue to restore a 3′-hydroxyl group; and
j) iteratively carrying out steps a) to i) to obtain the sequence of the nucleic acid template,
thereby sequencing the nucleic acid;
wherein the set of nucleotide analogues in step b)ii) comprises four nucleotide analogues, wherein the first nucleotide analogue has a first type of anchor moiety attached to the base via a first type of cleavable linker, the second nucleotide analogue has a second type of anchor moiety attached to the base via the first type of cleavable linker, the third nucleotide analogue has the second type of anchor moiety attached to the base via a second type of cleavable linker, and the fourth nucleotide analogue has the first type of anchor moiety attached to the base via the second type of cleavable linker.