Inhibiting denaturation of nucleic acid duplexes
AssigneeWhatman, Inc.
InventorsNeil Butt, Matthew Baker, Navin Pathirana
Life of this patent
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Abstract
A solution for treating a nucleic acid duplex having a pH of from 3 to 11, comprising a soluble protein or mixture of proteins; and 0.1 mM to 10 mM divalent cations; wherein the nature and concentration of the protein or mixture of proteins is selected so that the solution is capable of inhibiting heat denaturation of a nucleic acid duplex.
Claims29
A method for isolating nucleic acid which comprises:
- (a) applying a sample comprising cells containing nucleic acid to a filter, whereby the cells are retained as a retentate and contaminants are removed;
- (b) lysing the retentate from step (a) whilst the retentate is retained by the filter to form a cell lysate containing the nucleic acid;
- (c) filtering the cell lysate with the filter to retain the nucleic acid and remove remaining cell lysate;
- (d) washing the nucleic acid retained by the filter with a solution having a pH of from 3 to 11 and comprising (1) a soluble protein or mixture of proteins and (2) 0.1 mM to 10 mM divalent cations, wherein the nature and concentration of the protein or mixture of proteins is selected so that the solution is capable of inhibiting heat denaturation of a nucleic acid duplex;
- (e) heating the nucleic acid to an elevated temperature whilst retained by the filter;
- and
- (f) eluting the nucleic acid,
- wherein the filter composition and dimensions are selected so that the filter is capable of retaining the cells and the nucleic acid.
A kit for isolating nucleic acid from a sample comprising cells containing nucleic acid, comprising:
- (a) a filter supported by a support;
- (b) a solution having a pH of from 3 to 11 and comprising (1) a soluble protein or mixture of proteins and (2) 0.1 mM to 10 mM divalent cations, wherein the nature and concentration of the protein or mixture of proteins is selected so that the solution is capable of inhibiting heat denaturation of a nucleic acid duplex.
Use of a solution having a pH of from 3 to 11 and comprising (a) a soluble protein or mixture of proteins and (b) 0.1 mM to 10 mM divalent cations, wherein the nature and concentration of the protein or mixture of proteins is selected so that the solution is capable of inhibiting heat denaturation of a nucleic acid duplex, for inhibiting heat denaturation of a nucleic acid duplex and extracting double-stranded DNA from a biological sample comprising cells.
A Northern or Southern blotting method comprising (i) a step of inhibiting heat denaturation of a nucleic acid duplex, by contacting it with a solution comprising (a) a soluble protein or mixture of proteins and (b) 0.1 mM to 10 mM divalent cations, wherein the nature and concentration of the protein or mixture of proteins is selected so that the solution is capable of inhibiting heat denaturation of a nucleic acid duplex, prior to (ii) a step of heating the nucleic acid duplex to an elevated temperature.
Dependencies are inferred from the claim text.