US5073483A
Expired / Lapsed
US
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Method of staining nucleic acids detected by non-radioactive labelling and kit of reactants for carrying out the method
Assignee: BIOPROBE SYSTEMS 33 RUE de la BRECHE AUX LOUPS F-75012 PARIS (FRANCE)
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Filing: 1989-03-24
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Granted: 1991-12-17
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Expires: 2009-03-24
Abstract
Abstract A method of staining a nucleic acid sequence which is fixed on a solid support is described wherein an enzymatic system which fixes this sequence is employed to detect it by non-radioactive labelling, the enzymatic system being subsequently reacted with chromogenic substrate to produce a colored product. A developer solution is used comprising a sulfur organic compound constituting the substrate which is adapted to be hydrolized by the enzyme to form an organic thiol, and a metallic compound, for example a gold compound. The metallic compound is soluble and stable in aqueous solution and can react with the organic thiol to form a metal-organic compound complex having a characteristic color which precipitates in situ, which metal-organic complex subsequently may be decolorized with a reducing agent to thus allow reuse of the solid support. A set, or kit, of reactants for carrying out the method also is disclosed.
Inventors
- 👤 Philippe Lebacq
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-31 00:52
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/26; C12Q1/34; C12Q1/42
Claims Analysis
Total Claims: 17
Claim 1
Independent Claim
165 words
In a method for colorimetric detection of a nucleic acid sequence wherein the nucleic acid sequence is fixed on a solid support and an enzymatic detection system is sued to detect the nucleic acid sequence by non-isotopic labelling, the improvement comprising reversibly colorimetrically staining the non-isotopically labelled nucleic acid sequence on the solid support by the sequential steps of: (a) reacting the non-isotopically labelled nucleic acid sequence with a developer solution comprising a sulfur-containing organic compound containing a bond with a sulfur atom which constitutes a substrate for the enzymatic system and is hydrolyzed by said enzymatic system to form an organic thiol; (b) reacting with the organic thiol a metallic compound which is soluble and stable in aqueous solution to form a metal-sulfur compound complex having a characteristic color which precipitates in situ and which may subsequently be decolorized; and (c) decolorizing the colorimetrically stained labelled nuclear acid sequence with a reducing agent for the metal-sulfur complex whereby the solid support may be reused.
Claim 2
Dependent Claim
165 words
In a method for colorimetric detection of a nucleic acid sequence wherein the nucleic acid sequence is fixed on a solid support and an enzymatic detection system is sued to detect the nucleic acid sequence by non-isotopic labelling, the improvement comprising reversibly colorimetrically staining the non-isotopically labelled nucleic acid sequence on the solid support by the sequential steps of: (a) reacting the non-isotopically labelled nucleic acid sequence with a developer solution comprising a sulfur-containing organic compound containing a bond with a sulfur atom which constitutes a substrate for the enzymatic system and is hydrolyzed by said enzymatic system to form an organic thiol; (b) reacting with the organic thiol a metallic compound which is soluble and stable in aqueous solution to form a metal-sulfur compound complex having a characteristic color which precipitates in situ and which may subsequently be decolorized; and (c) decolorizing the colorimetrically stained labelled nuclear acid sequence with a reducing agent for the metal-sulfur complex whereby the solid support may be reused.
Claim 3
Dependent Claim
25 words
Method according to claim 1 wherein the reducing agent for the metal-sulfur complex is a solution consisting essentially of an acidic mixture of thiosulfate salts.
Claim 4
Dependent Claim
23 words
Method according to claim 2 wherein said decolorizing solution comprises a high concentration of sodium thiosulfate and ammonium thiosulfate in an acidic medium.
Claim 5
Dependent Claim
46 words
Method according to claim 3 wherein following treatment with said decolorizing solution said support is treated with a hot mixture of urea and sodium dodecyl sulfate to eliminate all traces of proteins and nucleic acids except for the nucleic acid sequence fixed on said solid support.
Claim 6
Dependent Claim
25 words
Method according to claim 1 wherein the metal of said metallic compound is selected from the group consisting of gold, mercury, silver, platinum and lead.
Claim 7
Dependent Claim
27 words
Method according to claim 5 wherein said metallic compound is a gold compound which can react with said organic thiol to form a yellow gold-sulfur compound complex.
Claim 8
Dependent Claim
24 words
Method according to claim 1 wherein said metal-sulfur compound complex is subsequently reacted with an intensifying compound to accentuate the staining of said complex.
Claim 9
Dependent Claim
46 words
Method according to claim 1 wherein said enzymatic system comprises a phosphatase enzyme comprising an alkaline phosphatase, and said developer solution comprises a phosphatase substrate, selected from the group consisting of cystamine-S-phosphate, adenine mononucleotide phosphate and adenosine-5'-monophosphate, and a metallic organic compound or a metallic salt.
Claim 10
Dependent Claim
36 words
Method according to claim 8 wherein said metallic compound is a gold compound, said enzymatic system comprises alkaline phosphatase and said developer solution comprises cystamine-S-phosphate constituting said phosphatase substrate and aurothioglucose constituting said metallic organic compound.
Claim 11
Dependent Claim
27 words
Method according to claim 9 wherein said developer solution comprises: ______________________________________
Tris-acetate 50 mM pH: 9.5
magnesium acetate 10 mM
cystamine-S-phosphate
5 mM
aurothioglucose 3 mM
______________________________________
Claim 12
Dependent Claim
33 words
Method according to claim 1 wherein said enzymatic system comprises the enzyme glutathion reductase and wherein said developer solution comprises a glutathion reductase substrate and a metallic organic compound or a metallic salt.
Claim 13
Dependent Claim
44 words
Method according to claim 11 wherein said metallic compound is a gold compound and said developer solution comprises: ______________________________________
Tris-HCl 50 mM pH: 7
nicotinamide adenine
5 mM
dinucleotide phosphate
(reduced form)
oxidized glutathion
5 mM
(sodium salts)
HAuCl.sub.4 or 5 mM
aurothioglucose
______________________________________
Claim 14
Dependent Claim
30 words
A method according to claim 7 wherein said intensifying compound is a silver salt, comprising silver nitrate, which can react with said metal-sulfur compound complex to produce a brown-black stain.
Claim 15
Dependent Claim
152 words
Set of reactants for staining a nucleic acid sequence fixed on a solid support wherein an enzymatic system has been used to detect said sequence by nonisotopic labelling by the method of claim 16, comprising: a container of a sulfur-containing organic compound containing a bond with a sulfur atom and forming a substrate for said enzymatic system, said sulfur organic compound being adapted to be hydrolyzed by said enzymatic system to produce an organic thiol, a container of a metallic compound which is soluble and stable in aqueous solution and which can react with said organic thiol to produce a metal-sulfur compound complex which precipitates in situ and has a characteristic color, a container of buffer solution which can be mixed with said substrate and metallic compound to form a developer solution, and a container of decolorizing solution consisting essentially of an aqueous solution of a reducing agent for the metal-sulfur complex.
Claim 16
Dependent Claim
30 words
Set of reactants according to claim 14 further comprising: a container of a solution of stain intensifying compound, and a container of a solution of urea and sodium dodecyl sulfate.
Claim 17
Dependent Claim
25 words
A set of reactants of claim 14 wherein the container of decolorizing solution is a solution consisting essentially of an acidic mixture of thiosulfate salts.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1994-12-16 | 1995-06-17 | 1995-12-17 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1998-12-16 | 1999-06-17 | 1999-12-17 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2002-12-16 | 2003-06-17 | 2003-12-17 | $7700.00 | Window Closed / Lapsed |