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US 9,670,243 B2Granted
Compositions and methods for sequencing nucleic acids
AssigneeIndustrial Technology Research Institute
InventorsChung-Fan Chiou, Chao-Chi Pan, Jenn-Yeh Fann, Shang-Chia Chang, Tsu-Ju Fu
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Abstract
Embodiments relate to methods of sequencing nucleic acids. Embodiments encompass the use of nucleotide analogs and a nucleic acid polymerase enzyme or enzyme complex comprising proofreading activity. The nucleotide analogs may become incorporated into a replicating strand and induce the proofreading activity of the polymerizing enzyme, thereby prolonging the duration of a signal associated with nucleotide incorporation, resulting in more observable sequencing events and increasing the accuracy of nucleic acid sequencing.
Claims28
A method of determining the nucleotide sequence of a target nucleic acid sequence, comprising the steps of
- (a) providing a reaction complex comprising a template nucleic acid comprising a target nucleic acid sequence, a primer nucleic acid comprising a. sequence which is complementary to a region of the template nucleic acid, and a polymerase enzyme or an enzyme complex, which comprises 5′ to 3′ polymerization activity and 3′ to 5′ exonuclease activity;
- (b) contacting the reaction complex with a plurality of nucleotide analogs, Wherein at least one nucleotide analog of said plurality comprises at least one base-pairing moiety, at least one non-complementary nucleotide residue and at least one label moiety comprising a photo-detectable label that is indicative of the identity of the base-pairing moiety, further wherein the at least one nucleotide analog of said plurality is chosen from compounds having formula I:
- or a pharmaceutically acceptable salt or hydrate thereof, wherein
- n is 1, 2, 3, 4, 5, 6, 7, 8, or 9;
- R1 and each R2 are O−;
- or R1 is
- and each R2 is O−;
- or R1 is O−, one R2, is
- and any remaining R2 is independently O−, S−, BH3−, or CH3;
- R3 is chosen from:
- wherein:
- B2 is chosen from adenine, cytosine, guanine, thymine, uracil, hypoxanthine, and 5-methylcytosine;
- X1 is chosen from methylene;
- L2;
- a base which does not base pair with any of adenine, cytosine, guanine, thymine, and uracil;
- and groups comprising L2 and a base which does not base pair with any of adenine, cytosine, guanine, thymine, and uracil;
- wherein L2 is chosen from alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, ester, amino, and sulfonyl;
- X2 is chosen from H, CH3, and a base which does not base pair with any of adenine, cytosine, guanine, thymine, and uracil;
- each R5 is independently H, OH, fluorine, or OCH3;
- Y2 is chosen from O−, S−, BH3−, and CH3
- F is a fluorescent dye;
- R4 is H, OH, halogen, alkyl (both substituted and unsubstituted), or alkoxy (both substituted and unsubstituted);
- Y1, and Y3 are each independently chosen from O−, S−, BH3−, and CH3 ;
- L1 is chosen from alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, ester, amino, and sulfonyl;
- Q is a fluorescence quenching moiety;
- and
- B1 is chosen from adenine, cytosine, guanine, thymine, uracil, hypoxanthine, and 5-methylcytosine;
- (c) allowing the enzyme or enzyme complex to incorporate a nucleotide analog in a template-dependent manner into a nascent strand via the enzyme's or enzyme complex's 5′ to 3′ polymerization activity, whereby the label moiety of the nucleotide analog is coupled to the nascent strand;
- (d) detecting the photo-detectable label of the incorporated nucleotide analog;
- (e) allowing the enzyme or enzyme complex to remove the label. moiety of the incorporated nucleotide analog from the nascent strand via the enzyme's or enzyme complex's 3′ to 5′ exonuclease activity;
- and
- (f) repeating steps (c)-(e) to determine the sequence of the target nucleic acid sequence.
29 more elements
A method of determining a nucleotide base incorporated by a polymerase enzyme or enzyme complex in a nucleic acid polymerization reaction, the method comprising the steps of:
- (a) conducting a nucleic acid polymerization reaction that utilizes both 5′ to 3′ polymerization activity and 3 to 5′ exonuclease activity of a polymerase enzyme or enzyme complex, and that results in production of a nascent strand in a template-dependent manner, Wherein said reaction is conducted in the presence of
- (i) a template nucleic acid comprising a target nucleic acid sequence,
- (ii) a primer nucleic acid comprising a sequence which is complementary to a region of the template nucleic acid,
- (iii) a polymerase enzyme or an enzyme complex comprising 5′ to 3′ polymerization activity and 3′ to 5′ exonuclease activity,
- (iv) a plurality of nucleotide analogs, wherein at least one nucleotide analog of said plurality comprises at least one base-pairing moiety, at least one non complementary nucleotide residue, and at least one label moiety, said label moiety comprising a photo-detectable label, wherein the at least one nucleotide analog of said plurality is chosen from compounds having formula I:
- or a pharmaceutically acceptable salt or hydrate thereof, wherein
- n is 1, 2, 3, 4, 5, 6, 7, 8, or 9;
- R1 and each R2 are O−;
- or R1 is
- and each R2 is O−;
- or R1 is O−, one R2 is
- and any remaining R2 is independently O−, S−, BH3−, or CH3;
- R3 is chosen from:
- wherein;
- B2 is chosen from adenine, cytosine, guanine, thymine, uracil, hypoxanthine, and 5-methylcytosine;
- X1 is chosen from methylene;
- L2;
- a base which does not base pair with any of adenine, cytosine, guanine, thymine, and uracil;
- and groups comprising L2 and a base which does not base pair with any of adenine, cytosine, guanine, thymine, and uracil, wherein L2 is chosen from alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, ester, amino, and sulfonyl;
- X2 is chosen from H, CH3, and a base which does not base pair with any of adenine, cytosine, guanine, thymine, and uracil;
- each R5 is independently H, OH, fluorine, or OCH3;
- Y2 is chosen from O−, S−, BH3−, and CH3
- F is a fluorescent dye;
- R4 is H, OH, halogen, alkyl (both substituted and unsubstituted), or alkoxy (both substituted and unsubstituted);
- Y1, and Y3 are each independently chosen from O−, S−, BH3−, and CH3 ;
- L1 is chosen from alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, ester, amino, and sulfonyl;
- Q is a fluorescence quenching moiety;
- and
- B1 is chosen from adenine, cytosine, guanine, thymine, uracil, hypoxanthine, and 5-methylcytosine,
- wherein the label moiety of an individual nucleotide analog becomes incorporated into the nascent strand and the 3′ to 5′ exonuclease activity of the enzyme or enzyme complex is allowed to remove the label moiety from the nascent strand;
- and
- (b) detecting the photo-detectable label, wherein said label is indicative of the identity of the base or bases present in the nucleotide analog incorporated by the enzyme or enzyme complex into the nascent strand.
29 more elements
A method of determining the nucleotide sequence of a target nucleic, acid sequence, comprising the steps of:
- (a) providing a reaction complex comprising a template nucleic acid comprising a target nucleic acid sequence, a primer nucleic acid comprising a sequence which is complementary to a. region of the template nucleic acid, and a polymerase enzyme or an enzyme complex, which comprises 5′ to 3′ polymerization activity and 3′ to 5′ exonuclease activity;
- (b) contacting the reaction complex with a plurality of nucleotide analogs;
- (c) allowing the enzyme or enzyme complex to incorporate a nucleotide analog in a template-dependent manner into a nascent strand via 5′ to 3′ polymerization activity of the enzyme or enzyme complex;
- (d) detecting the photo-detectable label moiety of the incorporated nucleotide analog;
- (e) allowing the enzyme or enzyme complex to remove the label moiety of the incorporated nucleotide analog from the nascent strand via 3′ to 5′ exonuclease activity of the enzyme or enzyme complex;
- and
- (f) repeating steps (c)-(e) to determine the sequence of the target nucleic acid sequence,
- wherein at least one of the nucleotide analogs is chosen from compounds having formula I:
- wherein
- n is 1, 2, 3, 4, 5, 6, 7, 8, or 9;
- R1 and each R2 are O−;
- or R1 is
- and each R2 is O−;
- or R1 is O−, one R2 is
- and any remaining R2 is independently O−, S−, BH3−, or CH3 ;
- R3 is chosen from:
- wherein
- B is chosen from adenine, cytosine, guanine, thymine, uracil, hypoxanthine, and 5-methylcytosine;
- X1 is chosen from methylene;
- L2;
- a base which does not base pair with any of adenine, cytosine, guanine, thymine, and uracil;
- and group comprising L2 and a base which does not base pair with any of adenine, cytosine, guanine, thymine, and uracil;
- wherein L1 is chosen from alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, ester, amino, and sulfonyl;
- X2 is chosen from H, CH3, and a base which does not base pair with any of adenine, cytosine, guanine, thymine, and uracil;
- each R5 is independently H, OH, fluorine, or OCH3;
- and
- Y2 is chosen from O−, S−, BH3−, and CH3 ;
- and
- F is a fluorescent dye;
- R4 is H, OH, halogen, alkyl (both substituted and unsubstituted), or alkoxy (both substituted and unsubstituted);
- Y1, and Y3 are each independently chosen from O−, S−, BH3−, and CH3 ;
- L1 is chosen from alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, ester, amino, and sulfonyl;
- Q is a fluorescence quenching moiety;
- and
- B1 is chosen from adenine, cytosine, guanine, thymine, uracil, hypoxanthine, and 5-methylcytosine.
32 more elements
Dependencies are inferred from the claim text.