Fluorescence energy transfer-based single molecule/ensemble dna sequencing by synthesis

A method for determining the nucleotide sequence of a single-stranded DNA comprising:

• a) contacting the single-stranded DNA which has a primer hybridized to a portion thereof, a polymerase, and 4 nucleotide analogues having the base of A, T, C and G, respectively, under conditions permitting the polymerase to form a ternary complex with the single-stranded DNA having the primer hybridized thereto and one of the nucleotide analogues, wherein said nucleotide analogue has a base that is complementary to a nucleotide residue of the single-stranded DNA which is immediately 5′ to a nucleoside residue of the single-stranded DNA hybridized to the 3′ terminal nucleotide residue of the primer, wherein said nucleotide analogue is not incorporated by the polymerase onto the primer, and wherein each nucleotide analogue has multiple fluorescent dyes attached thereto;
• b) identifying the nucleotide analogue in the ternary complex by detecting its unique fluorescent emission;
• c) contacting the ternary complex with 3′-O blocked nucleotide reversible terminators under conditions permitting the nucleotide polymerase to catalyze incorporation onto the primer of a 3′-O-blocked nucleotide reversible terminator complementary to a nucleotide residue of the single-stranded DNA which is immediately 5′ to a nucleotide residue of the single-stranded DNA hybridized to the 3′ terminal nucleotide residue of the primer, thereby replacing the nucleotide analogue in the ternary complex;
• d) cleaving the 3′-O-blocking moiety of the incorporated 3′-O blocked nucleotide reversible terminator, thereby resulting in a 3′-OH;
• and
• e) iteratively performing steps a) through c) for each nucleotide residue of the single-stranded DNA to be sequenced so as to thereby determine the sequence of the single-stranded DNA.

A real-time method for determining the nucleotide sequence of a single-stranded DNA comprising:

• a) contacting a composition comprising a single-stranded DNA which has a primer hybridized to a portion thereof, a nucleotide polymerase, and four nucleotide analogues having the structure:
• wherein the base is A, G, C, T, or U, or analogues thereof, wherein n is 0, 1, 2, 3, or 4, wherein the acceptor dye is one or more fluorophores,
• under conditions permitting the nucleotide polymerase to incorporate a nucleotide analogue when the nucleotide analogue has a base that is complementary to a nucleotide residue of the single-stranded DNA which is immediately 5′ to a nucleotide residue of the single-stranded DNA hybridized to the 3′ terminal nucleotide residue of the primer, and wherein the nucleotide polymerase has attached or incorporated fluorescence donor molecules,
• wherein the nucleotide polymerase has attached, incorporated, and/or conjugated fluorescence donor molecules, wherein the donor molecules are Forster Resonance Energy Transfer (FRET) donors, and the acceptor dyes and the acceptor dyes on the nucleotide analogue are corresponding FRET acceptors;
• b) exciting the nucleotide polymerase fluorescence donor molecules using an appropriate spectral emission, thereby causing the corresponding FRET acceptors attached to the nucleotide analogues, to generate the unique predetermined fluorescent wavelength emission, and thereby determine the identity of the nucleotide analogue;
• c) iteratively performing steps a) through b) for each nucleotide residue of the single-stranded DNA to be sequenced so as to thereby determine the sequence of the single-stranded DNA.

A method for determining the nucleotide sequence of a single-stranded DNA comprising:

• a) contacting a composition comprising a single-stranded DNA, wherein the single-stranded DNA has a primer hybridized to a portion thereof, with a non-catalytic metal ion, a nucleotide polymerase, and a nucleotide analogue having the structure:
• wherein the base is A, G, C, T, or U, or analogues thereof, wherein n is 0, 1, 2, 3, or 4, wherein the acceptor dye is one or more fluorophores, wherein R′ is a cleavable linker bound to a blocking moiety, and wherein cleaving the linker results in a 3′-OH,
• wherein the nucleotide polymerase has attached, incorporated, and/or conjugated fluorescence donor molecules, wherein the donor molecules are Forster Resonance Energy Transfer (FRET) donors, and the acceptor dyes on the nucleotide analogue are corresponding FRET acceptors, wherein (i) the type of base on each analogue is different from the type of base on each of the other three analogues, (ii) the fluorophores of each nucleotide analogue have a unique predetermined fluorescent wavelength emission that corresponds to the type of base, and (iii) the fluorophores of each analogue are FRET acceptors that are excited by same donor fluorescence molecules in the nucleotide polymerase;
• under conditions permitting the nucleotide polymerase to form a ternary complex with the single-stranded DNA, primer, and a nucleotide analogue wherein the nucleotide analogue has a base that is complementary to a nucleotide residue of the single-stranded DNA which is immediately 5′ to a nucleoside residue of the single-stranded DNA hybridized to the 3′ terminal nucleotide residue of the primer;
• b) exciting the nucleotide polymerase fluorescence donor molecules using an appropriate spectral emission, thereby causing the corresponding FRET acceptors, which are the acceptor dye fluorophores attached to the nucleotide analogue in the ternary complex, to generate the unique predetermined fluorescent wavelength emission, and thereby determine the identity of the nucleotide analogue;
• c) contacting the ternary complex with catalytic metal ions permitting the nucleotide polymerase to incorporate the nucleotide analogue into the primer;
• d) cleaving the linker bound to the blocking moiety of the incorporated analogue thereby resulting in a 3′-OH;
• e) iteratively performing steps a) through d) for each nucleotide residue of the single-stranded DNA to be sequenced so as to thereby determine the sequence of the single-stranded DNA.