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US 9,540,688 B2Granted
Real-time sequencing method for single molecule
AssigneePersonal Genomics, Inc.
InventorsTsu-Ju Fu, Shang-Chia Chang, Jenn-Yeh Fann
Life of this patent
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Abstract
This invention is to describe a method for the determination of a nucleic acid sequence, in which it involves an enzyme, an enzyme complex or plural number of enzymes with more than one enzymatic activity and a set of nucleotide analogs, to achieve high signal readout accuracy in nucleic acid sequencing by making each signal to have a long signaling time span which allows a higher signal clarity.
Claims9
A method of using nucleotide analogs to determine a nucleotide sequence of a target nucleic acid, comprising the steps of:
- (a) providing a reaction complex comprising a target nucleic acid, a primer nucleic acid comprising a sequence which is complementary to a region of the target nucleic acid, and a nucleic acid-polymerizing enzyme, enzyme complex, or enzyme mixture that possesses a 5′ to 3′ polymerization activity and a 3′ catalytic editing activity;
- (b) contacting the reaction complex with nucleotide analogs, wherein each nucleotide analog comprises a base moiety and at least one label moiety comprising a photo-detectable label, wherein the photo-detectable label is connected to a 3′ position of a sugar of the nucleotide analog via a coupler group, and the at least one label moiety includes a linker group selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, polyethylene glycol, ester, sulfonyl or a combination thereof, connecting the photo-detectable label to the coupler group, and the nucleotide analog has a structure as shown in Formula I or Formula II:
- wherein m is an integer and 1≦m≦10, L represents the linker group;
- Rm and Rm+1 vary as m varies, and when m is larger than 1, Rm refers to a set of functional groups ranging from R1 to Rm, each of Rm may represent independently hydrogen (H) or a functional group consisting of a linker group Lm and a quencher Qm, while Rm+1 may represent hydrogen (H) or a functional group consisting of a linker group Lm+1 and the quencher Qm+1, the quencher Qm or Qm+1 is independently chosen and applied to decrease the fluorescent signal generated from the fluorescent dye at the 3′-end of the nucleotide;
- the linker group Lm or Lm+1 is independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, polyethylene glycol, ester, amino, sulfonyl, or a combination thereof;
- F represents a photo-detectable label;
- B represents a base selected from adenine, cytosine, guanine, thymine, uracil, hypoxanthine or 5-methylcytosine;
- and
- Y in Formula I represents oxygen (O) or sulfur (S), while Y in Formula II represents methyl (—CH3) or boryl (—BH2);
- (c) allowing the nucleic acid-polymerizing enzyme, enzyme complex, or enzyme mixture to incorporate one nucleotide analog to the primer nucleic acid to form a nascent primer strand via the 5′ to 3′ polymerization activity of the nucleic acid-polymerizing enzyme, enzyme complex, or enzyme mixture, whereby the at least one label moiety and the base moiety of the nucleotide analog are incorporated in the nascent primer strand;
- (d) detecting the photo-detectable label of the at least one label moiety and determining an identity of the base moiety of the incorporated nucleotide analog;
- (e) removing the at least one label moiety of the incorporated nucleotide analog from the nascent primer strand via the 3′ catalytic editing activity of the nucleic acid-polymerizing enzyme, enzyme complex, or enzyme mixture;
- and
- (f) repeating steps (c)-(e) to determine the nucleotide sequence of the target nucleic acid.
11 more elements
A method of using nucleotide analogs to determine a nucleotide sequence of a target nucleic acid, comprising the steps of:
- (a) providing a reaction complex comprising a target nucleic acid, a primer nucleic acid comprising a sequence which is complementary to a region of the target nucleic acid, and a nucleic acid-polymerizing enzyme, enzyme complex, or enzyme mixture that possesses a 5′ to 3′ polymerization activity and a 3′ catalytic editing activity;
- (b) contacting the reaction complex with nucleotide analogs, wherein each nucleotide analog comprises a base moiety and at least one label moiety comprising a photo-detectable label, wherein the photo-detectable label is connected to a 3′ position of a sugar of the nucleotide analog via a coupler group, and the at least one label moiety includes a linker group selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, polyethylene glycol, ester, sulfonyl or a combination thereof, connecting the photo-detectable label to the coupler group, and the nucleotide analog has a structure as shown in Formula XVI:
- wherein m is an integer and 1≦m≦10, L represents the linker group;
- Rm and Rm+1 vary as m varies, and when m is larger than 1, Rm refers to a set of functional groups ranging from R1 to Rm, each of Rm may represent independently hydrogen (H) or a functional group consisting of a linker group Lm and a quencher Qm, while Rm+1 may represent hydrogen (H) or a functional group consisting of a linker group Lm+1 and the quencher Qm+1, the quencher Qm or Qm+1 is independently chosen and applied to decrease the fluorescent signal generated from the fluorescent dye at the 3′-end of the nucleotide;
- the linker group Lm or Lm+1 is independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, polyethylene glycol, ester, amino, sulfonyl, or a combination thereof;
- F represents a photo-detectable label;
- and
- B represents a base selected from adenine, cytosine, guanine, thymine, uracil, hypoxanthine or 5-methylcytosine;
- (c) allowing the nucleic acid-polymerizing enzyme, enzyme complex, or enzyme mixture to incorporate one nucleotide analog to the primer nucleic acid to form a nascent primer strand via the 5′ to 3′ polymerization activity of the nucleic acid-polymerizing enzyme, enzyme complex, or enzyme mixture, whereby the at least one label moiety and the base moiety of the nucleotide analog are incorporated in the nascent primer strand;
- (d) detecting the photo-detectable label of the at least one label moiety and determining an identity of the base moiety of the incorporated nucleotide analog;
- (e) removing the at least one label moiety of the incorporated nucleotide analog from the nascent primer strand via the 3′ catalytic editing activity of the nucleic acid-polymerizing enzyme, enzyme complex, or enzyme mixture;
- and
- (f) repeating steps (c)-(e) to determine the nucleotide sequence of the target nucleic acid.
10 more elements
A method of using nucleotide analogs to determine a nucleotide sequence of a target nucleic acid, comprising the steps of:
- (a) providing a reaction complex comprising a target nucleic acid, a primer nucleic acid comprising a sequence which is complementary to a region of the target nucleic acid, and a nucleic acid-polymerizing enzyme, enzyme complex, or enzyme mixture that possesses a 5′ to 3′ polymerization activity and a 3′ catalytic editing activity;
- (b) contacting the reaction complex with nucleotide analogs, wherein each nucleotide analog comprises a base moiety and at least one label moiety comprising a photo-detectable label, wherein the photo-detectable label is connected to a 3′ position of a sugar of the nucleotide analog via a coupler group, and the at least one label moiety includes a linker group selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, polyethylene glycol, ester, sulfonyl or a combination thereof, connecting the photo-detectable label to the coupler group, and the nucleotide analog has a structure as shown in Formula IA or Formula IIA:
- wherein m is an integer and 1≦m≦10, L represents the linker group;
- Rm and Rm+1 vary as m varies, and when m is larger than 1, Rm refers to a set of functional groups ranging from R1 to Rm, each of Rm may represent independently hydrogen (H) or a functional group consisting of a linker group Lm and a quencher Qm, while Rm+1 may represent hydrogen (H) or a functional group consisting of a linker group Lm+1 and the quencher Qm+1, the quencher Qm or Qm+1 is independently chosen and applied to decrease the fluorescent signal generated from the fluorescent dye at the 3′-end of the nucleotide;
- the linker group Lm or Lm+1 is independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, polyethylene glycol, ester, amino, sulfonyl, or a combination thereof;
- X represents the coupler group selected from —O—, —S—, —HN—, —COO—, —CO—, —NH(CS)NH—, or —NHCO—;
- F represents a photo-detectable label;
- B represents a base selected from adenine, cytosine, guanine, thymine, uracil, hypoxanthine or 5-methylcytosine;
- and
- Y in Formula I represents oxygen (O) or sulfur (S), while Y in Formula II represents methyl (—CH3) or boryl (—BH2);
- (c) allowing the nucleic acid-polymerizing enzyme, enzyme complex, or enzyme mixture to incorporate one nucleotide analog to the primer nucleic acid to form a nascent primer strand via the 5′ to 3′ polymerization activity of the nucleic acid-polymerizing enzyme, enzyme complex, or enzyme mixture, whereby the at least one label moiety and the base moiety of the nucleotide analog are incorporated in the nascent primer strand;
- (d) detecting the photo-detectable label of the at least one label moiety and determining an identity of the base moiety of the incorporated nucleotide analog;
- (e) removing the at least one label moiety of the incorporated nucleotide analog from the nascent primer strand via the 3′ catalytic editing activity of the nucleic acid-polymerizing enzyme, enzyme complex, or enzyme mixture;
- and
- (f) repeating steps (c)-(e) to determine the nucleotide sequence of the target nucleic acid.
12 more elements
Dependencies are inferred from the claim text.