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

A plurality of different deoxyribonucleic acids covalently immobilized on a plurality of spots on a solid surface, a) wherein a plurality of a first deoxyribonucleic acid on a first spot of the solid surface comprises a DNA polymerase-incorporated labeled deoxyribonucleotide analogue derived from the group consisting of labeled deoxyribonucleotide analogues A-D;

• b) wherein a plurality of a second deoxyribonucleic acid on a second spot of the solid surface comprises a DNA polymerase-incorporated labeled deoxyribonucleotide analogue derived from the group consisting of deoxyribonucleotide analogues derived from A-D, wherein the DNA polymerase-incorporated labeled deoxyribonucleotide analogue in b) is different from the DNA polymerase-incorporated labeled deoxyribonucleotide analogue in a);
• and c) wherein a plurality of a third deoxyribonucleic acid on a third spot of the solid surface comprises a DNA polymerase-incorporated un-labeled deoxyribonucleotide analogue 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 a 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 a growing DNA strand during a 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.