US5620852A
Expired / Lapsed
US
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Nucleic acid preparation methods
Assignee: Cerus Corp
•
Filing: 1994-10-31
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Granted: 1997-04-15
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Expires: 2014-10-31
Abstract
Abstract The present invention provides a much simplified method for the preparation of nucleic acid samples. This method provides many advantages over prior sample preparation methods in terms of greater sensitivity, reliability and ease of use. Importantly, the present invention provides a method in which nucleic acid samples may be prepared so as to conserve reagents and time. The samples so prepared are readily amplifiable and may be used for other purposes as well.
Inventors
- 👤 Lily Lin
- 👤 George Cimino
- 👤 Yu S. Zhu
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 13:48
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/1003; C12Q; C12Q1/00; C12Q1/68; C12Q1/6806
Claims Analysis
Total Claims: 22
Claim 1
Independent Claim
96 words
A method for preparing nucleic acid samples comprising, in sequential order, the steps of: a) providing in any order, i) a plasma sample suspected of containing ribonucleic acid, ii) an aqueous buffered solution consisting essentially of guanidinium thiocyanate, and beta-mercaptoethanol, iii) an alcohol, and iv) a reaction vessel; b) adding to said reaction vessel, in any order, said sample and said aqueous buffered solution, to make a reaction mixture; c) heating said reaction mixture to a temperature above 60° C.; d) adding said alcohol to precipitate said ribonucleic acid; and e) recovering said precipitated ribonucleic acid.
Claim 2
Independent Claim
96 words
A method for preparing nucleic acid samples comprising, in sequential order, the steps of: a) providing in any order, i) a plasma sample suspected of containing ribonucleic acid, ii) an aqueous buffered solution consisting essentially of guanidinium thiocyanate, and beta-mercaptoethanol, iii) an alcohol, and iv) a reaction vessel; b) adding to said reaction vessel, in any order, said sample and said aqueous buffered solution, to make a reaction mixture; c) heating said reaction mixture to a temperature above 60° C.; d) adding said alcohol to precipitate said ribonucleic acid; and e) recovering said precipitated ribonucleic acid.
Claim 3
Dependent Claim
10 words
The method of claim 1 wherein said alcohol is isopropanol.
Claim 4
Dependent Claim
21 words
The method of claim 1 further comprising the step of cooling said reaction mixture after step (c) prior to step (d).
Claim 5
Dependent Claim
17 words
The method of claim 3 wherein said cooling is performed until the reaction mixture reaches room temperature.
Claim 6
Dependent Claim
18 words
The method of claim 3 wherein said cooling is performed until the reaction mixture reaches approximately 4° C.
Claim 7
Dependent Claim
13 words
The method of claim 1 wherein said ribonucleic acid is from a pathogen.
Claim 8
Dependent Claim
18 words
The method of claim 1 further comprising the step of amplifying said ribonucleic acid recovered in step (e).
Claim 9
Independent Claim
117 words
A method for preparing nucleic acid samples from human pathogens comprising, in sequential order, the steps of: a) providing in any order, i) a sample of human plasma suspected of containing the ribonucleic acid of a human pathogen, ii) an aqueous buffered solution consisting essentially of guanidinium thiocyanate and beta-mercaptoethanol, iii) isopropanol, and iv) a reaction vessel; b) adding to said reaction vessel, in any order, said sample and said aqueous buffered solution, to make a reaction mixture; c) heating said reaction mixture to a temperature of 65° C.; d) cooling said reaction mixture to a temperature of approximately 4° C.; e) adding said isopropanol to precipitate said ribonucleic acid; and f) recovering said precipitated ribonucleic acid.
Claim 10
Independent Claim
117 words
A method for preparing nucleic acid samples from human pathogens comprising, in sequential order, the steps of: a) providing in any order, i) a sample of human plasma suspected of containing the ribonucleic acid of a human pathogen, ii) an aqueous buffered solution consisting essentially of guanidinium thiocyanate and beta-mercaptoethanol, iii) isopropanol, and iv) a reaction vessel; b) adding to said reaction vessel, in any order, said sample and said aqueous buffered solution, to make a reaction mixture; c) heating said reaction mixture to a temperature of 65° C.; d) cooling said reaction mixture to a temperature of approximately 4° C.; e) adding said isopropanol to precipitate said ribonucleic acid; and f) recovering said precipitated ribonucleic acid.
Claim 11
Dependent Claim
18 words
The method of claim 8 further comprising the step of amplifying said ribonucleic acid recovered in step (f).
Claim 12
Dependent Claim
30 words
The method of claim 9 wherein said amplifying of said ribonucleic acid is performed by adding a thermostable DNA polymerase having endogenous reverse transcriptase activity to said recovered ribonucleic acid.
Claim 13
Independent Claim
122 words
A method for preparing nucleic acid samples comprising, in sequential order, the steps of: a) providing in any order, i) a plasma sample suspected of containing ribonucleic acid, ii) an aqueous buffered solution consisting essentially of guanidinium thiocyanate and beta-mercaptoethanol, iii) isopropanol, and iv) a reaction vessel; b) adding to said reaction vessel, in any order, said sample and said aqueous buffered solution; c) heating said reaction mixture to a temperature of approximately 65° C.; d) cooling said reaction mixture to a temperature of approximately 4° C.; e) adding said isopropanol to said reaction mixture in the approximate proportion of one part reaction mixture to at least one part isopropanol to precipitate said ribonucleic acid; and f) recovering said precipitated ribonucleic acid.
Claim 14
Independent Claim
122 words
A method for preparing nucleic acid samples comprising, in sequential order, the steps of: a) providing in any order, i) a plasma sample suspected of containing ribonucleic acid, ii) an aqueous buffered solution consisting essentially of guanidinium thiocyanate and beta-mercaptoethanol, iii) isopropanol, and iv) a reaction vessel; b) adding to said reaction vessel, in any order, said sample and said aqueous buffered solution; c) heating said reaction mixture to a temperature of approximately 65° C.; d) cooling said reaction mixture to a temperature of approximately 4° C.; e) adding said isopropanol to said reaction mixture in the approximate proportion of one part reaction mixture to at least one part isopropanol to precipitate said ribonucleic acid; and f) recovering said precipitated ribonucleic acid.
Claim 15
Dependent Claim
14 words
The method of claim 11 wherein said target ribonucleic acid is from a pathogen.
Claim 16
Dependent Claim
18 words
The method of claim 11 further comprising the step of amplifying said ribonucleic acid recovered in step (f).
Claim 17
Dependent Claim
30 words
The method of claim 7 wherein said amplifying of said ribonucleic acid is performed by adding a thermostable DNA polymerase having endogenous reverse transcriptase activity to said recovered ribonucleic acid.
Claim 18
Dependent Claim
15 words
The method of claim 7 further comprising the step of detecting said amplified ribonucleic acid.
Claim 19
Dependent Claim
13 words
The method of claim 8 wherein said human pathogen is a viral pathogen.
Claim 20
Dependent Claim
13 words
The method of claim 16 wherein said viral pathogen is Hepatitis C Virus.
Claim 21
Dependent Claim
12 words
The method of claim 12 wherein said pathogen is a viral pathogen.
Claim 22
Dependent Claim
30 words
The method of claim 13 wherein said amplifying of said ribonucleic acid is performed by adding a thermostable DNA polymerase having endogenous reverse transcriptase activity to said recovered ribonucleic acid.
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