COMPOSITIONS AND METHODS FOR ARRAY-BASED NUCLEIC ACID HYBRIDIZATION
The invention provides compositions and methods for generating a molecular profile of genomic DNA by hybridization of labeled nucleic acid representing the genomic DNA to immobilized nucleic acid probes, e.g., arrays or biochips.
1 - 66 . (canceled)
67 . A method for hybridizing a sample of labeled nucleic acid targets to a plurality of nucleic acid probes, comprising the following steps:
(a) providing a sample of nucleic acid targets comprising fluorescent-labeled nucleic acid fragments, wherein the fluorescent label is sensitive to oxidation and wherein the nucleic acid fragments have a size less than 200 bases;
(b) providing a plurality of nucleic acid probes; and
(c) contacting the fragmented, labeled nucleic acid targets and the plurality of nucleic acid probes under conditions allowing hybridization of the sample with the probes, wherein the hybridization conditions comprise use of a hybridization solution comprising at least one antioxidant, wherein the amount of antioxidant in the solution is sufficient to inhibit the oxidation of the fluorescent label under the hybridization conditions.
68 . The method of claim 67 , wherein the fluorescent label comprises Cy5™.
69 . The method of claim 67 , wherein the fluorescent dye comprises a rhodamine, a fluorescein or an aryl-substituted 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene dye.
70 . The method of claim 67 , wherein the antioxidant is present in the hybridization solution at a concentration of 25 mM to 1000 mM.
71 . The method of claim 70 , wherein the antioxidant is present in the hybridization solution at a concentration of 50 mM to 500 mM
72 . The method of claim 67 , wherein the antioxidant comprises a mercapto-containing compound.
73 . The method of claim 72 , wherein the mercapto-containing compound comprises a 2-mercaptoethylamine, a thiol N-acetylcysteine, an ovothiol, or a 4-mercaptoimidazole.
73 . The method of claim 67 , wherein the antioxidant comprises an antioxidant vitamin-containing compound.
74 . The method of claim 73 , wherein the antioxidant vitamin-containing compound comprises an ascorbic acid (Vitamin C) or a tocopherol (Vitamin E).
75 . The method of claim 67 , wherein the antioxidant comprises a propyl gallate.
76 . The method of claim 67 , wherein the antioxidant comprises a beta-carotene.
77 . The method of claim 67 , wherein the antioxidant comprises a butylated hydroxytoluene (BHT) or a butylated hydroxyanisole (BHA).
78 . The method of claim 67 , in which the size of the fragments is no more than 175 bases.
79 . The method of claim 67 , in which the size of the fragments is no more than 150 bases.
80 . The method of claim 67 , in which the size of the fragments is no more than 125 bases.
81 . The method of claim 67 , in which the size of the fragments is no more than 100 bases.
82 . The method of claim 67 , in which the size of the fragments is no more than 75 bases.
83 . The method of claim 67 , in which the size of the fragments is 25 to 30 bases.
84 . A kit comprising a sample of fluorescent dye-labeled nucleic acid in a solution comprising at least one antioxidant and printed matter, wherein the printed matter comprises instructions on using the fluorescent dye-labeled nucleic acid in a hybridization reaction with another nucleic acid, and wherein the sample of fluorescent dye-labeled nucleic acid comprises nucleic acid fragments having a size no more than 200 bases.
85 . The kit of claim 84 , further comprising a hybridization complex wash solution comprising at least one antioxidant.
86 . The kit of claim 84 , wherein the fluorescent dye comprises Cy5™.
87 . The kit of claim 84 , wherein the fluorescent dye comprises a rhodamine, a fluorescein or an aryl-substituted 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene dye.
88 . The kit of claim 84 , in which the fragments having a size no more than 175 bases.
89 . The kit of claim 84 , in which the size of the fragments is no more than 150 bases.
90 . The kit of claim 84 , in which the size of the fragments is no more than 125 bases.
91 . The kit of claim 84 , in which the size of the fragments is no more than 100 bases.
92 . The kit of claim 84 , in which the size of the fragments is no more than 75 bases.
93 . The kit of claim 84 , in which the size of the fragments is 25 to 30 bases.
94 . A composition comprising a sample of Cy5™-labeled nucleic acid in a solution comprising at least one antioxidant, wherein the Cy5™-labeled nucleic acid sample comprises fragments having a size no more than 200 bases.