COMPOSITIONS AND METHODS FOR NUCLEIC ACID SEQUENCING
Embodiments of the present application relates to methods and compositions for increasing fluorescent dye signal intensity during sequencing by synthesis. In particular, the compositions and methods described herein involve the use aqueous scan mixtures including one or more water-soluble macrocycles that can form host-guest complexes with the dyes.
1 . A method of sequencing a plurality of different target polynucleotides, comprising:
(a) contacting a solid support with an incorporation mixture comprising DNA polymerase and one or 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;
(b) incorporating one type of nucleotides into the sequencing primers to produce extended copy polynucleotides, wherein one or more of the four types of nucleotides comprises a detectable label; and
(c) imaging and performing one or more fluorescent measurements of the extended copy polynucleotides in an aqueous scan mixture;
wherein the aqueous scan mixture comprises one or more additives for enhancing fluorescent signal intensity of the detectable label, and wherein the one or more additives comprise one or more water-soluble macrocycles.
2 . The method of claim 1 , wherein the water-soluble macrocycle forms a host-guest complex with the detectable label.
3 . The method of claim 1 , wherein the water-soluble macrocycle comprises water-soluble cyclodextrins, water-soluble calixarenes, water-soluble cucurbiturils, or optionally substituted analogs, salts, or hydrates thereof, or combination thereof.
4 . The method of claim 3 , wherein the water-soluble cyclodextrins or the optionally substituted analogs, salts, or hydrates thereof comprise α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or substituted analogs, salts or hydrates thereof, or combination thereof.
5 . The method of claim 3 , wherein the substituted analogs of the water-soluble cyclodextrins are independently substituted with one or more substituents selected from the group consisting of sulfonate, sulfo, hydroxy, carboxy, carboxylate, succinyl, C 1 -C 6 alkyl, C 1 -C 6 alkyl substituted with sulfo, sulfonate, carboxy, carboxylate, or hydroxy, a hydroxy protecting group, —C(═O) (C 1 -C 6 alkyl), —C(═O) CH 3 , and —C(═O) Ph, and combinations thereof.
6 . The method of claim 3 , wherein the water-soluble cyclodextrin or the substituted analogs, salts, or hydrates thereof are selected from the group consisting of α-cyclodextrin, β-cyclodextrin, (2-hydroxypropl)-β-cyclodextrin, acetyl-β-cyclodextrin, (2-hydroxyethyl)-β-cyclodextrin, heptakis (2,3,6-tri-O-methyl)-β-cyclodextrin, succinyl-β-cyclodextrin, methyl-β-cyclodextrin, carboxymethyl-β-cyclodextrin, heptakis (6-deoxy-6-amino)-β-cyclodextrin heptahydrochloride, γ-cyclodextrin hydrate, (2-hydroxypropyl)-γ-cyclodextrin, and salts and hydrates thereof, and combinations thereof.
7 . The method of claim 6 , wherein the water-soluble cyclodextrin is (2-hydroxyporpyl)-β-cyclodextrin.
8 . The method of claim 6 , wherein the water-soluble cyclodextrin is methyl-β-cyclodextrin.
9 . The method of claim 3 , wherein the concentration of the water-soluble cyclodextrin or the optionally substituted analog, salt, or hydrate thereof in the aqueous scan mixture is from about 1 mM to about 500 mM.
10 . The method of claim 3 , wherein the binding affinity constant of the water-soluble cyclodextrin to the detectable label is at least about 500M −1 .
11 . The method of claim 3 , wherein the water-soluble cucurbituril or the substituted analogs, salts, or hydrates thereof comprise cucurbituril hydrates or substituted analogs or salts thereof, or combination thereof.
12 . The method of claim 11 , wherein cucurbituril hydrates are selected from the group consisting of cucuribit [5]uril hydrate, cucuribit [6]uril hydrate, cucuribit [7]uril hydrate, and cucuribit [8]uril hydrate.
13 . (canceled)
14 . The method of claim 3 , wherein the substituted analogs of the water-soluble cucurbiturils are independently substituted with one or more substituents selected from the group consisting of sulfonate, sulfo, hydroxy, carboxy, carboxylate, succinyl, C 1 -C 6 alkyl, C 1 -C 6 alkyl substituted with sulfo, sulfonate, carboxy, carboxylate, or hydroxy, a hydroxy protecting group, —C(═O) (C 1 -C 6 alkyl), —C(═O) CH 3 , and —C(═O) Ph, and combinations thereof.
15 . The method of claim 3 , wherein the binding affinity constant of the water-soluble cucurbituril to the detectable label is at least about 1×10 5 M −1 .
16 . The method of claim 3 , wherein the concentration of the water-soluble cucurbituril or the optionally substituted analog, salt, or hydrate thereof in the aqueous scan mixture is from about 0.01 mM to about 1 mM.
17 . The method of claim 3 , wherein the water-soluble calixarenes or the optionally substituted analogs, salts, or hydrates thereof comprise sulfocalixarenes or substituted analogs, salts or hydrates thereof, or combination thereof.
18 . The method of claim 3 , wherein the substituted analogs of the water-soluble calixarenes are independently substituted with one or more substituents selected from the group consisting of sulfonate, sulfo, hydroxy, carboxy, carboxylate, succinyl, C 1 -C 6 alkyl, C 1 -C 6 alkyl substituted with sulfo, sulfonate, carboxy, carboxylate, or hydroxy, a hydroxy protecting group, —C(═O) (C 1 -C 6 alkyl), —C(═O) CH 3 , and —C(═O) Ph, and combinations thereof.
19 . The method of claim 3 , wherein the water-soluble calixarenes or the optionally substituted analogs, salts, or hydrates thereof are selected from the group consisting of 4-sulfocalix [4] arene, 4-sulfocalix [4] arene hydrate, 4-sulfothiacalix [4] arene, and salts and combinations thereof.
20 . The method of claim 3 , wherein the binding affinity constant of the water-soluble calixarene to the detectable label is at least about 1000M −1 .
21 . The method of claim 3 , wherein the concentration of the water-soluble calixarene or the optionally substituted analog, salt, or hydrate thereof in the aqueous scan mixture is from about 0.01 mM to about 10 mM.
22 .- 23 . (canceled)
24 . A method for enhancing the fluorescence of a fluorescent dye, comprising:
contacting the fluorescent dye with an aqueous scan mixture composition comprising one or more water-soluble macrocycles, wherein the water-soluble macrocycle forms a host-guest complex with the fluorescent dye.
25 .- 36 . (canceled)
37 . A kit for use with a sequencing apparatus, comprising:
an incorporation mixture comprising one or more different types of nucleotides, wherein at least one nucleotide is labeled with a detectable label;
an aqueous scan mixture comprising one or more water-soluble macrocycles, wherein the water-soluble macrocycle is capable of encapsulating the detectable label to form a host-guest complex with the detectable label.
38 .- 50 . (canceled)