Patent ID: 11952624
Assignee: GENESENSE TECHNOLOGY INC., SHANGHAI (CN)
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 0:
1. A method for sequencing a nucleic acid molecule, comprising:
(1) providing a nucleic acid molecule to be sequenced that is linked to a support, or Linking a nucleic acid molecule to be sequenced to a support;
(2) adding a primer for initiating a nucleotide polymerization reaction, a polymerase for performing the nucleotide polymerization reaction, and four compounds to form a reaction system containing a solution phase and a solid phase;
wherein, the four compounds are derivatives of nucleotides A, (T/U), C and G, respectively, each of which including a nitrogenous base, and have the ability of base complementary pairing;
wherein the hydroxyl (—OH) at the 3′-position of ribose or deoxyribose of each of the four compounds is protected by a reversible protecting group;
each of the four compounds takes the general formula of NT-L1-L2-Lb, where NT denotes a nucleotide of A, T(U), C, and G, L1 denotes a first linker, L2 denotes a second linker, and Lb denotes a terminal molecular label binding to or reactive to a receptor in a detectable group comprising a luminescence-activating molecule and the receptor, the luminescence-activating molecule capable of causing emission of fluorescence when bound to a suitable substrate without being excited by external photoexcitation;
wherein the reversible protecting group and the first linker of each of the four compounds both includes a functional group which can be cleaved at a same reaction condition;
wherein the second linker L2 in each of the four compounds are different, denoted as L2A, L2T(U), L2C and L2G, respectively, and at least three of the second linkers are each cleavable under a condition under which the second linker in the remaining compounds as well as the first linker are not cleaved; wherein the second linker in one of the four compounds can be optionally absent;
(3) annealing the primer to the nucleic acid molecule to be sequenced, and forming a duplex linked to the support by using the primer as an initial growing nucleic acid chain together with the nucleic acid molecule to be sequenced;
(4) using the polymerase to carry out the nucleotide polymerization reaction under a condition that allows the polymerase to carry out the nucleotide polymerization reaction, thereby incorporating one of the four compounds into the 3′-end of the growing nucleic acid chain;
(5) allowing the duplex to contact the detectable group to thereby cause a coupling reaction or specific binding between the molecular label of the incorporated compound and the receptor in the detectable group, and allowing the luminescence-activating molecule to contact the substrate to undergo a fluorescence reaction, and detecting a first fluorescence signal;
(6) carrying out a first cleaving reaction at a condition suitable to cleave the second linker of a first one of L2A, L2T(LT), L2C and L2G, and detecting the presence or absence of a second fluorescent signal;
(7) carrying out a second cleaving reaction at a condition suitable to cleave the second linker of a second one of L2A, L2T(U), L2C and L2G, and detecting the presence or absence of a third fluorescent signal;
(8) carrying out a third cleaving reaction at a condition suitable to cleave the second linker of a third one of L2A, L2T(U), L2C and L2G, and detecting the presence or absence of a fourth fluorescent signal;
(9) based on the pattern of the detected presence or absence of the first, second, third, and fourth fluorescent signal, determining the identity of the nucleotide of the incorporated compound to be one of A, (T/U), C and G; and
(10) cleaving the first linker of the incorporated compound while removing the protecting group at the 3′-position of the ribose or deoxyribose of the incorporated compound at a suitable reaction condition and to recover the nitrogenous base of the incorporated compound.