US4830969A
Expired / Lapsed
US
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Process for the rapid and simple isolation of nucleic acids
Assignee: Research Foundation of the State University of New York
•
Filing: 1981-08-31
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Granted: 1989-05-16
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Expires: 2001-08-31
Abstract
Abstract A process for the separation from other cellular materials of heat agglomeration resistant water soluble nitrogen containing organic compounds such as plasmids, RNA's, mitochondrial DNA's, viral DNA's, chloroplast DNA's, other episomal DNA's and certain proteins. The process comprises heating cellular materials in a solution of lysing agent to lyse the desired cells and to agglomerate water soluble nitrogen containing compounds such as certain chromosomal DNA's which are not resistant to agglomeration; centrifuging the resulting product to remove water soluble agglomerated materials; separating the supernatant liquid and precipitating the water soluble agglomeration resistant organic compounds with a water soluble precipitant. The process also includes separating the agglomeration resistant water soluble nitrogen containing compounds from each other by means of exclusion chromotography.
Inventors
- 👤 David S. Holmes
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-28 11:42
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/1003; C12P; C12P19/00; C12P19/26; C12P19/28; C12P19/30; C12P19/34; Y; Y10; Y10S; Y10S435/00; Y10S435/82; Y10S530/00; Y10S530/82
Claims Analysis
Total Claims: 37
Claim 1
Independent Claim
108 words
A process for the separation of agglomeration resistant water soluble nitrogen containing cellular organic compounds, selected from the group consisting of water soluble agglomeration resistant nucleic acids, proteins and peptides, from other cellular materials, which process comprises: heating cellular material in a solution of lysing agent to a temperature between 60° and 105° C. for between 10 and 180 seconds to lyse the desired cells and to agglomerate water soluble nitrogen containing compounds which are not resistant to agglomeration; centrifuging the resulting product to remove water insoluble and agglomerated materials; separating the supernatant liquid; and precipitating the water soluble agglomeration resistant organic compounds with a water soluble precipitant.
Claim 2
Dependent Claim
108 words
A process for the separation of agglomeration resistant water soluble nitrogen containing cellular organic compounds, selected from the group consisting of water soluble agglomeration resistant nucleic acids, proteins and peptides, from other cellular materials, which process comprises: heating cellular material in a solution of lysing agent to a temperature between 60° and 105° C. for between 10 and 180 seconds to lyse the desired cells and to agglomerate water soluble nitrogen containing compounds which are not resistant to agglomeration; centrifuging the resulting product to remove water insoluble and agglomerated materials; separating the supernatant liquid; and precipitating the water soluble agglomeration resistant organic compounds with a water soluble precipitant.
Claim 3
Dependent Claim
14 words
The process of claim 1 wherein the agglomeration resistant organic compounds comprises a protein.
Claim 4
Dependent Claim
30 words
The process of claim 1 wherein the agglomeration resistant organic compounds comprise a nucleic acid selected from the group consisting of plasmids, RNA's, mitochondrial DNA's, viral DNA's and chloroplast DNA's.
Claim 5
Dependent Claim
12 words
The process of claim 3 wherein the nucleic acid comprises a plasmid.
Claim 6
Dependent Claim
11 words
The process of claim 3 wherein the nucleic acid comprises RNA.
Claim 7
Dependent Claim
14 words
The process of claim 1 wherein the water soluble precipitant is isopropanol or ethanol.
Claim 8
Dependent Claim
14 words
The process of claim 4 wherein the water soluble precipitant is isopropanol or ethanol.
Claim 9
Dependent Claim
14 words
The process of claim 5 wherein the water soluble precipitant is isopropanol or ethanol.
Claim 10
Dependent Claim
11 words
The process of claim 1 wherein the lysing agent comprises lysozyme.
Claim 11
Dependent Claim
11 words
The process of claim 4 wherein the lysing agent comprises lysozyme.
Claim 12
Dependent Claim
11 words
The process of claim 5 wherein the lysing agent comprises lysozyme.
Claim 13
Dependent Claim
26 words
The process of claim 1 wherein the product is centrifuged at from between 3000 to about 30,000 G's for from about 3 to about 15 minutes.
Claim 14
Dependent Claim
41 words
The process of claim 1 wherein the precipitated agglomeration resistant water soluble organic compounds contain plasmids and RNA; the precipitate is separated from the supernatant liquid, resuspended in another liquid, and the plasmids are separated from the RNA by exclusion chromatography.
Claim 15
Dependent Claim
20 words
The process of claim 13 wherein the RNA is fragmented prior to being separated from the plasmids by exclusion chromotography.
Claim 16
Dependent Claim
25 words
The process of any of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 wherein the lysing agent comprises a surfactant.
Claim 17
Dependent Claim
26 words
The process of any of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 wherein the lysing agent comprises a chelating agent.
Claim 18
Dependent Claim
33 words
The process of any of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 wherein the lysing agent comprises a buffer to maintain the pH between 5 and 10.
Claim 19
Dependent Claim
54 words
The process of claim 1 wherein the precipitated agglomeration resistant water soluble organic compounds contain plasmids and RNA; the precipitate is separated from the supernatant; the precipitate is resuspended in an aqueous liquid to which density gradient chemicals are added and the plasmids are separated from RNA and residual DNA by density gradient centrifuging.
Claim 20
Dependent Claim
33 words
The process of claim 1 wherein the precipitated agglomeration resistant water soluble organic compounds are separated from the supernatant liquid; resuspended in an aqueous liquid and treated with phenol to remove residual DNA.
Claim 21
Dependent Claim
125 words
A process for the separation of agglomeration resistant water soluble nitrogen containing cellular organic compounds, selected from the group consisting of water soluble agglomeration resistant nucleic acids, proteins and peptides, from other cellular materials, which process comprises: heating cellular material in a solution of lysing agent comprising at least one of urea or guanidinium hydrochloride to a temperature of between about 60° and about 105° C. for between about 10 and about 180 seconds to lyse the desired cells and to agglomerate water soluble nitrogen containing compounds which are not resistant to agglomeration; centrifugating the resulting product to remove water insoluble and agglomerated materials; separating the supernatant liquid; and, precipitating from said liquid the water soluble agglomeration resistant organic compounds with a water soluble precipitant.
Claim 22
Dependent Claim
125 words
A process for the separation of agglomeration resistant water soluble nitrogen containing cellular organic compounds, selected from the group consisting of water soluble agglomeration resistant nucleic acids, proteins and peptides, from other cellular materials, which process comprises: heating cellular material in a solution of lysing agent comprising at least one of urea or guanidinium hydrochloride to a temperature of between about 60° and about 105° C. for between about 10 and about 180 seconds to lyse the desired cells and to agglomerate water soluble nitrogen containing compounds which are not resistant to agglomeration; centrifugating the resulting product to remove water insoluble and agglomerated materials; separating the supernatant liquid; and, precipitating from said liquid the water soluble agglomeration resistant organic compounds with a water soluble precipitant.
Claim 23
Dependent Claim
11 words
The process of claim 20 wherein the lysing agent comprises urea.
Claim 24
Dependent Claim
12 words
The process of claim 20 wherein the lysing agent comprises guanidinium hydrochloride.
Claim 25
Dependent Claim
30 words
The process of claim 20 wherein the agglomeration resistant organic compounds comprise a nucleic acid selected from the group consisting of plasmids, RNA's, mitochondrial DNA's, viral DNA's and chloroplast DNA's.
Claim 26
Dependent Claim
12 words
The process of claim 23 wherein the nucleic acid comprises a plasmid.
Claim 27
Dependent Claim
11 words
The process of claim 23 wherein the nucleic acid comprises RNA.
Claim 28
Dependent Claim
11 words
The process of claim 25 wherein the lysing agent comprises urea.
Claim 29
Dependent Claim
14 words
The process of claim 20 wherein the water soluble precipitant is isopropanol or ethanol.
Claim 30
Dependent Claim
13 words
The process of claim 20 wherein the lysing agent additionally comprises a surfactant.
Claim 31
Dependent Claim
14 words
The process of claim 20 wherein the lysing agent additionally comprises a chelating agent.
Claim 32
Dependent Claim
22 words
The process of claim 20 wherein the lysing agent additionally comprises a buffer to maintain pH between about 5 and about 10.
Claim 33
Dependent Claim
26 words
The process of claim 20 wherein the product is centrifuged at from between 3000 to about 30,000 G's for from about 3 to about 15 minutes.
Claim 34
Dependent Claim
41 words
The process of claim 20 wherein the precipitated agglomeration resistant water soluble organic compounds contain plasmids and RNA; the precipitate is separated from the supernatant liquid, resuspended in another liquid; and, the plasmids are separated from the RNA by exclusion chromotography.
Claim 35
Dependent Claim
20 words
The process of claim 32 wherein the RNA is fragmented prior to being separated from the plasmids by exclusion chromotography.
Claim 36
Dependent Claim
54 words
The process of claim 20 wherein the precipitated agglomeration resistant water soluble organic compounds contain plasmids and RNA; the precipitate is separated from the supernatant; the precipitate is resuspended in an aqueous liquid to which density gradient chemicals are added; and, the plasmids are separated from RNA and residual DNA by density gradient centrifuging.
Claim 37
Dependent Claim
33 words
The process of claim 20 wherein the precipitated agglomeration resistant water soluble organic compounds are separated from the supernatant liquid; resuspended in an aqueous liquid; and, treated with phenol to remove residual DNA.
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| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 1992-05-15 | 1992-11-14 | 1993-05-16 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1996-05-15 | 1996-11-14 | 1997-05-16 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2000-05-15 | 2000-11-14 | 2001-05-16 | $7700.00 | Window Closed / Lapsed |