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US 2022/0290227 A1Published application
Dna sequencing with non-fluorescent nucleotide reversible terminators and cleavable label modified nucleotide terminators
AssigneeThe Trustees of Columbia University in the City of New York
InventorsJingyue Ju, Dae Hyun Kim, Jia Guo, Qinglin Meng, Zengmin Li, Huanyan Cao
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Abstract
This invention provides a process for sequencing nucleic acids using 3′ modified deoxynucleotide analogues or 3′ modified deoxyinosine triphosphate analogues, and 3′ modified dideoxynucleotide analogues having a detectable marker attached thereto.
Claims28
A method for determining the identity of each of a series of consecutive nucleotide residues in a nucleic acid comprising:
- a) contacting a plurality of the nucleic acids with (i) a dideoxynucleotide triphosphate (ddNTP) analogue having the structure:
- wherein F is a fluorophore, L is a cleavable linker molecule and the base is adenine, guanine, cytosine, uracil or thymine, and wherein each base has a different fluorophore attached, and wherein the fluorophore attached to each type of base differs in its excitation or emission spectra from the fluorophores attached to the other types of bases, (ii) a deoxynucleotide triphosphate (dNTP) analogue having the structure:
- wherein B is a base and is adenine, guanine, cytosine, uracil, or thymine or is an inosine, and wherein R′ is a cleavable chemical group, (iii) a nucleic acid polymerase and (iv) at least two primers each of which hybridizes with a separate nucleic acid of the plurality of nucleic acids,
- under conditions permitting a ddNTP analogue that is complementary to the consecutive nucleotide residue to be identified to form a phosphodiester bond with the 3′ end of one of the primers and a dNTP analogue that is complementary to the consecutive nucleotide residue to be identified to form a phosphodiester bond with the 3′ end of another of the primers;
- b) identifying the fluorophore of the ddNTP analogue which has formed the phosphodiester bond thereby identifying the consecutive nucleotide;
- c) cleaving the fluorophore from the ddNTP analogue which has formed the phosphodiester bond and cleaving the cleavable chemical group from the dNTP which has formed the phosphodiester bond;
- d) iteratively repeating steps a) through c) for each of the consecutive nucleotide residues to be identified until the final consecutive nucleotide residue is to be identified;
- e) repeating steps a) and b) to identify the final consecutive nucleotide residue,
- thereby determining the identity of each of the series of consecutive nucleotide residues in the nucleic acid.
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A method for determining the identity of consecutive nucleotide residues in a self-priming nucleic acid comprising:
- a) contacting a plurality of the nucleic acids with (i) a dideoxynucleotide triphosphate (ddNTP) analogue having the structure:
- wherein F is a fluorophore, L is a cleavable linker molecule and the base is adenine, guanine, cytosine, uracil or thymine, wherein each base has a different fluorophore attached, (ii) a deoxynucleotide triphosphate (dNTP) analogue having the structure:
- wherein B is a base and is adenine, guanine, cytosine, uracil, thymine or an inosine, and wherein R′ is a cleavable chemical group, and (iii) a nucleic acid polymerase,
- under conditions permitting a ddNTP analogue that is complementary to the consecutive nucleotide residue to be identified to form a phosphodiester bond with the 3′ end of one of the self-priming nucleic acids and a dNTP analogue that is complementary to the consecutive nucleotide residue to be identified to form a phosphodiester bond with the 3′ end of another of the self-priming nucleic acids;
- b) identifying the fluorophore of the ddNTP analogue which has formed the phosphodiester bond thereby identifying the identify of the consecutive nucleotide;
- c) cleaving the fluorophore from the ddNTP analogue which has formed the phosphodiester bond and cleaving the cleavable chemical group from the dNTP which has formed the phosphodiester bond;
- d) iteratively repeating steps a) through c) for each of the consecutive nucleotide residues to be identified until the final consecutive nucleotide residue is to be identified;
- e) repeating steps a) and b) to identify the final consecutive nucleotide residue,
- thereby determining the identity of each of the series of consecutive nucleotide residues in the nucleic acid.
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A deoxyribonucleic acid having attached at a 3′ end thereof, by a phosphodiester bond, a compound having the structure:
- wherein the O atom labeled α is the 3′ O atom of the deoxyribonucleic acid, the wavy line represents the remainder of the deoxyribonucleic acid that is 5′ relative to the 3′O, and wherein R′ is a cleavable chemical group.
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Dependencies are inferred from the claim text.