Compositions and methods for reducing photo damage during sequencing
OwnerXiaolin WU, Benedict MACKWORTH +6inventors
InventorsXiaolin WU, Benedict MACKWORTH, Madushani DHARMARWARDANA, Patrick MCCAULEY, Philip STACKHOUSE, Xiaohai LIU, Tao YAN, Krystle REISS
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Abstract
Embodiments of the present disclosure relate to cyclooctatetraene containing dyes and their uses as fluorescent labels. Also provided are composition containing cyclooctatetraene. The dyes and compositions may be used in various biological applications, such as nucleic acid sequencing.
Claims37
A fluorescent compound excitable by a light source having a wavelength between about 400 nm to about 500 nm, wherein the fluorescent compound is covalently attached to one or more photoprotective moieties comprising the formula (Ia), (Ib), (IIa), (IIb), (III) or (IV):
- wherein
- each of R1, R3a, R3b, R5 and R6 is independently H, unsubstituted C1-C6 alkyl, or substituted C1-C6 alkyl;
- R2 is —OR7, —NR8R9, —O— or —NR8—;
- each of R4a and R4b is independently H or optionally substituted C6-C10 aryl;
- each of R7, R8 and R9 is independently H, unsubstituted C1-C6 alkyl, substituted C1-C6 alkyl, optionally substituted C6-C10 aryl, optionally substituted C3-C7 cycloalkyl, optionally substituted 5 to 10 membered heteroaryl, or optionally substituted 3 to 10 membered heterocyclyl;
- each of the aromatic ring or ring system in formulas (Ia), (Ib), (IIa), (IIb), (III) or (IV) is optionally substituted with one, two or three RAr independently selected from the group consisting of carboxyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C1-C6 halo alkyl, C1-C6 halo alkoxy, (C1-C6 alkoxy)C1-C6 alkyl, —O(C1-C6 alkoxy)C1-C6 alkyl, optionally substituted amino, amino(C1-C6 alkyl), halo, cyano, hydroxy, hydroxy(C1-C6 alkyl), nitro, sulfonyl, sulfo, sulfonate, S-sulfonamido, N-sulfonamido, C6-C10 aryl, 5 to 10 membered heteroaryl, C3-C10 carbocyclyl, 3 to 10 membered heterocyclyl, —C(═O)OR10, —C(═O)NR11R12;
- —(CH2CH2O)n(C1-C6 alkyl), and —O(CH2CH2O)n(C1-C6 alkyl);
- each of R10, R11 and R12 is independently H, unsubstituted C1-C6 alkyl, or substituted C1-C6 alkyl;
- and
- n is an integer of 1 to 20.
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-40. (canceled)
A method for reducing light-induced sequencing signal decay during sequencing by synthesis, comprising:
- (i) contacting a solid support with an incorporation mixture comprising DNA polymerase and one more of four different types of nucleotides, wherein the solid support comprises a plurality of different target polynucleotides immobilized thereon, and sequencing primers that are complementary and hybridized to at least a portion of the target polynucleotides;
- (ii) incorporating one type of nucleotides into the sequencing primers to produce extended copy polynucleotides, wherein one or more four types of nucleotides comprises a detectable label, and each of the four types of nucleotides comprises a 3′ blocking group;
- (iii) imaging and performing one or more fluorescent measurements of the extended copy polynucleotides in an aqueous scan mixture to determine the identity of the incorporated nucleotides;
- and
- (iv) removing the 3′ blocking groups and the detectable labels of the incorporated nucleotides;
- wherein the aqueous scan mixture comprises one or more additives for reducing fluorescent signal decay caused by the fluorescent measurements, and wherein the one or more additives comprise a tri-substituted 1,4-cyclooctatetraene (COT) analog.
-50 (canceled)
A kit for use with a sequencing apparatus, comprising a scan mixture composition, wherein the scan mixture composition comprises one or more additives for reducing fluorescent signal decay caused by the fluorescent measurements, and wherein the one or more additives comprise a tri-substituted 1,4-cyclooctatetraene (COT) analog.
-59. (canceled)
Dependencies are inferred from the claim text.