Compositions and methods for nucleotide sequencing
First Claim
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1. A method for sequencing a nucleic acid molecule, comprising:
- (a) contacting a nucleic acid template, a nucleic acid primer complementary to a portion of said template, an extension enzyme, and a reversibly blocked nucleotide under conditions wherein said primer is extended to incorporate said nucleotide into a sequence complementary to said nucleic acid template,said reversibly blocked nucleotide comprising a sugar moiety and a base moiety, wherein said base moiety is linked to a label by an enzymatically cleavable peptide linker, andwherein, attached at the 2′
or 4′
position of said sugar moiety, is a reversible blocking group comprising —
CH2—
CH═
CH2, —
CH2—
N3, —
CH2—
S—
S—
tBu, —
CH2—
S—
Me, —
CH2—
0—
CH2—
CH2—
CN, —
CH2—
CH2—
CN, —
C(O)—
CH3, —
CH3, 2-nitrobenzyl, tetrandyropyran, 4-nitrobenzoyl, 2-aminobenzoyl, —
C(O)-linker-dye, 2-nitrobenzyl-linker-dye, —
C(O)—
O—
CH2—
CH═
CH-linker-dye, —
NH—
C(O)—
CH3, —
F, —
NH2, phosphate, or sulfate,(b) identifying the nucleotide in said sequence complementary to said nucleic acid template;
(c1) enzymatically cleaving the peptide linker; and
(c2) removing the blocking group,wherein identifying said nucleotide identifies at least a portion of the sequence of said nucleic acid molecule.
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Abstract
The invention provides nucleoside and nucleotide molecules containing cleavable linkers linking a label such as a dye. The invention also provides nucleosides and nucleotide molecules containing a blocking group, either removable or non-removable. The invention additionally provides methods of using the nucleoside and nucleotide molecules containing a cleavable linker and/or a blocking group.
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Citations
25 Claims
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1. A method for sequencing a nucleic acid molecule, comprising:
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(a) contacting a nucleic acid template, a nucleic acid primer complementary to a portion of said template, an extension enzyme, and a reversibly blocked nucleotide under conditions wherein said primer is extended to incorporate said nucleotide into a sequence complementary to said nucleic acid template, said reversibly blocked nucleotide comprising a sugar moiety and a base moiety, wherein said base moiety is linked to a label by an enzymatically cleavable peptide linker, and wherein, attached at the 2′
or 4′
position of said sugar moiety, is a reversible blocking group comprising —
CH2—
CH═
CH2, —
CH2—
N3, —
CH2—
S—
S—
tBu, —
CH2—
S—
Me, —
CH2—
0—
CH2—
CH2—
CN, —
CH2—
CH2—
CN, —
C(O)—
CH3, —
CH3, 2-nitrobenzyl, tetrandyropyran, 4-nitrobenzoyl, 2-aminobenzoyl, —
C(O)-linker-dye, 2-nitrobenzyl-linker-dye, —
C(O)—
O—
CH2—
CH═
CH-linker-dye, —
NH—
C(O)—
CH3, —
F, —
NH2, phosphate, or sulfate,(b) identifying the nucleotide in said sequence complementary to said nucleic acid template; (c1) enzymatically cleaving the peptide linker; and (c2) removing the blocking group, wherein identifying said nucleotide identifies at least a portion of the sequence of said nucleic acid molecule. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
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Specification