Design and synthesis of cleavable fluorescent nucleotides as reversible terminators for dna sequencing by synthesis

A method for sequencing a DNA template of at least 24 nucleotides comprising detecting the identities of deoxyribonucleotide analogues of adenine, guanine, cytosine, and thymine incorporated into the end of a growing strand of complementary DNA synthesized using a DNA polymerase in a DNA polymerase reaction, wherein the deoxyribonucleotide analogues act as reversible terminators in the DNA polymerase reaction and wherein the reversible terminators comprise labeled and unlabeled deoxyribonucleotide analogues, a) wherein at least a first labeled deoxyribonucleotide analogue is derived from the group consisting of labeled deoxyribonucleotide analogues A-D:

• b) wherein at least a second labeled deoxyribonucleotide analogue is derived from the group consisting of labeled deoxyribonucleotide analogues A-D, wherein the second labeled deoxyribonucleotide analogue in b) is different from the first labeled deoxyribonucleotide analogue in a);
• and c) wherein at least one unlabeled deoxyribonucleotide analogue is derived from the group consisting of un-labeled deoxyribonucleotide analogues A′-D′:
• wherein R (a) represents a cleavable, chemical group capping the oxygen at the 3′ position of the deoxyribose of the deoxyribonucleotide analogue, (b) does not interfere with recognition of the deoxyribonucleotide analogue as a substrate by the DNA polymerase or with incorporation of the deoxyribonucleotide analogue into the growing DNA strand during the DNA polymerase reaction, and (c) is stable during the DNA polymerase reaction;
• wherein the covalent bond between the 3′-oxygen and R is stable during the DNA polymerase reaction;
• wherein the F on each of the deoxyribonucleotide analogues represents a distinguishable, detectable moiety;
• wherein L represents a cleavable, chemical linker which (a) does not interfere with recognition of the deoxyribonucleotide analogue as a substrate by the DNA polymerase or with incorporation of the deoxyribonucleotide analogue into the growing DNA strand during the DNA polymerase reaction and (b) is stable during the DNA polymerase reaction;
• wherein (i) the deoxyribonucleotide analogue is incorporated into the growing DNA strand as a result of the DNA polymerase reaction, (ii) the incorporated deoxyribonucleotide analogue is identified, and (iii) the cleavable chemical linker L, and the covalent bond between the 3′-oxygen and R are cleaved under conditions compatible with DNA sequencing to allow the incorporation and detection of the next deoxyribonucleotide analogue;
• wherein the deoxyribonucleotide analogue:
• i) is recognized as a substrate by the DNA polymerase for incorporation into the growing DNA strand during the DNA polymerase reaction, ii) is efficiently and accurately incorporated at the end of the growing DNA strand during the DNA polymerase reaction, iii) produces a 3′-OH group on the deoxyribose upon cleavage of R under conditions compatible with DNA sequencing, and iv) no longer includes the detectable moiety on the base upon cleavage of L under conditions compatible with DNA sequencing;
• and wherein if the deoxyribonucleotide analogue is:
• (A) or (A′), it forms hydrogen bonds with cytosine or a cytosine deoxyribonucleotide analogue;
• (B) or (B′), it forms hydrogen bonds with thymine or a thymine deoxyribonucleotide analogue;
• (C) or (C′), it forms hydrogen bonds with guanine or a guanine deoxyribonucleotide analogue;
• or (D) or (D′), it forms hydrogen bonds with adenine or an adenine deoxyribonucleotide analogue.