US5422241A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Methods for the recovery of nucleic acids from reaction mixtures
Assignee: Applied Biosystems LLC
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Filing: 1991-07-03
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Granted: 1995-06-06
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Expires: 2011-07-03
Abstract
Abstract A method for the recovery of nucleic acids from a reaction mixture, such as nuclease protection assays, comprising the use of one reagent containing a) nuclease for digesting single-stranded RNA and b) a nucleic acid precipitating carrier agent, (sheared DNA or linear acrylamide). A second reagent contains a chaotropic agent (a guanidinium salt) for inactivating said nucleases and an alcohol (ethanol or isopropanol) for the simultaneous inactivation of the nucleases and the precipitation of the nucleic acids without the need for protease digestion or organic extraction.
Inventors
- 👤 Marianna Goldrick
- 👤 Matthew Winkler
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 15:50
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/1003; C12Q; C12Q1/00; C12Q1/68; C12Q1/6806
Claims Analysis
Total Claims: 14
Claim 1
Independent Claim
116 words
A method for the recovery of nucleic acid from a reaction mixture comprising: preparing a reaction mixture comprising RNA, a nuclease effective to digest single-stranded RNA, and a nucleic acid precipitating carrier agent at a concentration effective to assist in the precipitation of undigested RNA; incubating the reaction mixture under conditions effecting enzymatic degradation of single-stranded nucleic acid; adding to the reaction mixture a reagent comprising a nuclease inactivating chaotropic agent in a concentration sufficient to inactivate the nuclease and a nucleic acid precipitating alcohol in a concentration sufficient to precipitate undigested RNA; and inactivating the nuclease while essentially simultaneously precipitating nucleic acids from the reaction mixture without organic extraction of proteins from the reaction mixture.
Claim 2
Independent Claim
116 words
A method for the recovery of nucleic acid from a reaction mixture comprising: preparing a reaction mixture comprising RNA, a nuclease effective to digest single-stranded RNA, and a nucleic acid precipitating carrier agent at a concentration effective to assist in the precipitation of undigested RNA; incubating the reaction mixture under conditions effecting enzymatic degradation of single-stranded nucleic acid; adding to the reaction mixture a reagent comprising a nuclease inactivating chaotropic agent in a concentration sufficient to inactivate the nuclease and a nucleic acid precipitating alcohol in a concentration sufficient to precipitate undigested RNA; and inactivating the nuclease while essentially simultaneously precipitating nucleic acids from the reaction mixture without organic extraction of proteins from the reaction mixture.
Claim 3
Dependent Claim
12 words
The method of claim 1 wherein the carrier agent comprises linear acrylamide.
Claim 4
Dependent Claim
12 words
The method of claim 1 wherein the carrier agent comprises DNA fragments.
Claim 5
Dependent Claim
13 words
The method of claim 1 wherein the chaotropic agent comprises a guanidinium compound.
Claim 6
Dependent Claim
15 words
The method of claim 4 wherein the guanidinium compound comprises guanidinium thiocyanate or guanidinium HCl.
Claim 7
Dependent Claim
29 words
The method of claim 1 wherein the added reagent further comprises a nuclease inactivation enhancing reducing agent in a concentration sufficient to assist in the inactivation of the nuclease.
Claim 8
Dependent Claim
13 words
The method of claim 6 wherein the reducing agent comprises mercaptoethanol or dithiothreitol.
Claim 9
Dependent Claim
28 words
The method of claim 1 wherein the added reagent further comprises a nuclease inactivation enhancing detergent in a concentration sufficient to promote in the inactivation of the nuclease.
Claim 10
Dependent Claim
16 words
The method of claim 8 wherein the nuclease inactivation enhancing detergent is SDS or N-lauroyl sarcosine.
Claim 11
Dependent Claim
13 words
The method of claim 1 wherein the alcohol comprises isopropyl alcohol or ethanol.
Claim 12
Dependent Claim
16 words
The method of claim 1 wherein the precipitated nucleic acid is separated by centrifugation or filtration.
Claim 13
Dependent Claim
20 words
The method of claim 1 wherein the reaction mixture comprises nuclease A, T1, S1 or RNase I from E. coli.
Claim 14
Dependent Claim
14 words
The methods of claim 12 wherein the reaction mixture comprises nuclease A or T1.
References Cited (Backward Citations)
US4843155A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1998-06-05 | 1998-12-05 | 1999-06-06 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2002-06-05 | 2002-12-05 | 2003-06-06 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2006-06-05 | 2006-12-05 | 2007-06-06 | $7700.00 | Window Closed / Lapsed |