COMPOSITIONS AND METHODS FOR PREPARING SHORT RNA MOLECULES AND OTHER NUCLEIC ACIDS
The invention provides methods of preparing nucleic acids, such as RNA molecules, of a defined size or range of sizes. The invention provides compositions, methods and kits for use in the production and preparation of small RNA molecules (including without limitation micro-RNA, siRNA, d-siRNA and e-siRNA) and other nucleic acids of various sizes.
1 - 62 . (canceled)
63 . A method for preparing nucleic acids of a preselected size comprising:
(a) adding a fluid mixture or, in any order, fluids or combinations of fluids that contain the components of said fluid mixture to a sample comprising nucleic acids of said preselected size and other nucleic acids, to produce a binding mixture;
(b) filtering said binding mixture through an affinity column, wherein nucleic acids of said preselected size and said other nucleic acids bind to a composition within the affinity column; and optionally washing the bound nucleic acids; and
(c) eluting the bound nucleic acids of said preselected size with a second fluid mixture, to produce an eluate, wherein nucleic acids of said preselected size are present in said eluate, and said other nucleic acids remain bound to the composition within said affinity column.
64 . The method of claim 63 , wherein at least one of said affinity columns comprises glass fibers.
65 . The method of claim 63 , wherein said nucleic acids of a preselected size are RNA molecules.
66 . The method of claim 63 , wherein one or more of said fluid mixtures comprises one or more alcohols.
67 . The method of claim 66 , wherein said alcohol is selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, isobutyl alcohol, tertiary butyl alcohol, 1-hexanol, 2-hexanol, and 3-hexanol.
68 . A method for preparing nucleic acids of a preselected size comprising:
(a) adding a fluid mixture or, in any order, fluids or combinations of fluids that contain the components of said fluid mixture to a sample comprising nucleic acids of a preselected size and other nucleic acids having a size other than said preselected size to produce a binding mixture;
(b) filtering said binding mixture through an affinity column and collecting the flow-through therefrom, wherein said nucleic acids of a preselected size are present in said flow-through, and said other nucleic acids remain bound to the composition within said affinity column.
69 . The method of claim 68 , wherein at least one of said affinity columns comprises glass fibers.
70 . The method of claim 68 , wherein said nucleic acids of a preselected size are RNA molecules.
71 . The method of claim 68 , wherein one or more of said fluid mixtures comprises one or more alcohols.
72 . The method of claim 71 , wherein said alcohol is selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, isobutyl alcohol, tertiary butyl alcohol, 1-hexanol, 2-hexanol, and 3-hexanol.
73 . A method for preparing nucleic acids of a preselected size comprising:
(a) adding a first fluid mixture or, in any order, fluids or combinations of fluids that contain the components of said first fluid mixture, to a sample comprising nucleic acids of a preselected size and other nucleic acids having a size other than said preselected size to produce a first binding mixture;
(b) filtering said first binding mixture through a first affinity column, wherein nucleic acids of said preselected size pass through a composition within the first affinity column, and said other nucleic acids are retained, to produce a first flow-through solution;
(c) adding a second fluid mixture or, In any order, fluids or combinations of fluids that contain the components of said second fluid mixture to said first flow-through solution, to produce a second binding mixture;
(d) filtering the second binding mixture through a second affinity column, wherein nucleic acids of said preselected size bind to a composition within the second affinity column and, optionally, washing the bound nucleic acids; and
(e) eluting nucleic acids of said preselected size with a third fluid mixture, to produce an eluate, wherein nucleic acids of said preselected size molecules are present in said eluate.
74 . The method of claim 73 , wherein at least one of said affinity columns comprises glass fibers.
75 . The method of claim 73 , wherein said nucleic acids of a preselected size are RNA molecules.
76 . The method of claim 73 , wherein one or more of said fluid mixtures comprises one or more alcohols.
77 . The method of claim 76 , wherein said alcohol is selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, isobutyl alcohol, tertiary butyl alcohol, 1-hexanol, 2-hexanol, and 3-hexanol.
78 . A method for preparing nucleic acids of a preselected size comprising:
(a) adding a first fluid mixture or, in any order, fluids or combinations of fluids that contain the components of said first fluid mixture, to a nuclease reaction mix, to produce a first binding mixture, wherein the first fluid mixture is essentially free of alcohol;
(b) filtering said first binding mixture through a first affinity column, wherein longer RNA molecules in the first binding mixture (i.e., RNA having a length of about “y” by to about “z” or more bp) bind to, and shorter RNA molecules (i.e., RNA having a length of about 5 bp to about “y” bp) pass through, a composition within said first affinity column, to produce a first flow-through solution comprising RNA having a length of about 5 bp to about “y” bp;
(c) eluting said longer RNA from said first affinity column, to produce a first eluate comprising longer RNA molecules having lengths in the range of about “y” by to about “z” or more bp;
(d) adding one or more alcohol(s) to said first flow-through solution to bring the final concentration to about 10% (v/v), to produce a second binding mixture;
(e) filtering said second binding mixture through a second affinity column, wherein longer RNA molecules in said second binding mixture (i.e., RNA having a length of about “x” by to about “y” or more bp) bind to, and shorter RNA molecules (i.e., RNA having a length of about 5 bp to about “x” bp) pass through, a composition within said second affinity column, to produce a second flow-through solution comprising RNA having a length of about 5 bp to about “x” bp;
(f) eluting the longer RNA from said second affinity column, to produce a second eluate comprising longer RNA molecules having lengths in the range of about “x” by to about “y” bp;
(g) adding one or more alcohol(s) to said second flow-through solution to bring the final concentration to about 10% (v/v) more than the first flow-through solution, to produce a third binding mixture;
(h) filtering said third binding mixture through a third affinity column, wherein longer RNA molecules in said second binding mixture (i.e., RNA having a length of about “w” by to about “x” bp) bind to, and shorter RNA molecules (i.e., RNA having a length of about 5 bp to about “w” bp) pass through a composition within said third affinity column, to produce a third flow-through solution comprising RNA having a length of about 5 bp to about “w” bp; and
(i) eluting the longer RNA from said second affinity column, to produce a third eluate comprising longer RNA molecules having lengths in the range of about “w” by to about “x” bp;
wherein “w”<“x”<“y”<“z”, and wherein said first eluate comprises RNA molecules having an average length greater than that of the RNA molecules in said second eluate, and said second eluate comprises RNA molecules having an average length greater than that of said RNA molecules in said third eluate.
79 . The method of claim 78 , wherein at least one of said affinity columns comprises glass fibers.
80 . The method of claim 78 , wherein said nucleic acids of a preselected size are RNA molecules.
81 . The method of claim 78 , wherein one or more of said fluid mixtures comprises one or more alcohols.