EP0245945A2
Expired / Lapsed
EP
⏱️ 0.0 years remaining
Automated nucleic acid extractor
Assignee: Applied Biosystems Inc
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Filing: 1987-04-08
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Granted: —
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Expires: 2007-04-08
Abstract
Abstract An automated apparatus is provided which implements a new method of extracting and purifying nucleic acids from cells without the use of centrifugation. In the method, a lysate is created by treating the cells with proteinase K in the presence of a lysis buffer having a high concentration of a salt. The lysate is mixed with a phenol-based solvent system, thereby creating an emulsion. The emulsion is heated to promote phase separation. Similarly, the rate of phase separation is also enhanced by increasing the surface area of the emulson. Once the phase separation is complete, the lower organic phase is removed and the upper aqueous phase is repeatedly extracted with the phenol-based solvent a preselected number of times, and is finally extracted using chloroform. The remaining aqueous phase is then dialyzed to further purify and concentrate the nucleic acid solution. Two preferred embodiments of apparatus are presented to accomplish this extraction.
Inventors
- 👤 Guy Richard Cathcart
- 👤 P. Eric Mayrand
- 👤 Eric S. Nordman
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-05 15:02
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/0004; C12N9/0012; B; B01; B01J; B01J19/00; B01J19/0046; C12M; C12M47/00; C12M47/06; C12N15/00; C12N15/09; C12N15/10; C12N15/1003; B01J2219/00; B01J2219/00274; B01J2219/00277; B01J2219/00279; B01J2219/00281; B01J2219/00286; B01J2219/00306; B01J2219/00308; B01J2219/00331; B01J2219/00351; B01J2219/00389; B01J2219/00479; B01J2219/00488; B01J2219/0068; B01J2219/00686; B01J2219/00689; B01J2219/00695; C40; C40B; C40B60/00; C40B60/14
Claims Analysis
Total Claims: 7
Claim 1
Independent Claim
152 words
An apparatus for extracting nucleic acids comprising: reaction vessel means for holding a sample to be extracted, said reaction vessel means being of a flow through type comprising a tube having an opening at one end for loading said sample, hereinafter the top end, and an opening at the other end, hereinafter the bottom end; rocker means for holding said reaction vessel means, and for rocking said reaction vessel means back and forth about a horizontal axis and for rotating said reaction vessel means about said horizontal axis; fluid delivery means for controlled delivery of proteinase K, a lysis buffer, a phenol based solvent, and chloroform to said reaction vessel means; waste manifold means for draining fluids from said reaction vessel means through said other end; and said reaction vessel means, said fluid delivery means, and said waste manifold means all mounted to said rocker means to be rotated and rocked together.
Claim 2
Independent Claim
152 words
An apparatus for extracting nucleic acids comprising: reaction vessel means for holding a sample to be extracted, said reaction vessel means being of a flow through type comprising a tube having an opening at one end for loading said sample, hereinafter the top end, and an opening at the other end, hereinafter the bottom end; rocker means for holding said reaction vessel means, and for rocking said reaction vessel means back and forth about a horizontal axis and for rotating said reaction vessel means about said horizontal axis; fluid delivery means for controlled delivery of proteinase K, a lysis buffer, a phenol based solvent, and chloroform to said reaction vessel means; waste manifold means for draining fluids from said reaction vessel means through said other end; and said reaction vessel means, said fluid delivery means, and said waste manifold means all mounted to said rocker means to be rotated and rocked together.
Claim 3
Dependent Claim
18 words
A device as in claim 1 wherein said other end of said reaction vessel comprises a Luer-type fitting.
Claim 4
Dependent Claim
45 words
A device as in claim 2 further comprising dialysis cartridge means having a Luer-type fitting for connecting to and mating with said Luer-type fitting on said reaction vessel and for receiving extracted nucleic acid from said reaction vessel means for dialysis in said dialysis cartridge.
Claim 5
Dependent Claim
40 words
A device as in claim 2 further comprising precipitate collection means for collecting nucleic acids precipitated in said reaction vessel means, said precipitate collection means having a Luer-type fitting for mating with said Luer-type fitting on said reaction vessel means.
Claim 6
Independent Claim
315 words
A method of extracting nucleic acids from a sample contained in a reaction vessel mounted on a rocker apparatus, said reaction vessel being of a flow through type having an opening on one end for receiving said sample and an opening on the other end connected to a valve for controlling eduction of fluids from said reaction vessel, said rocker apparatus having a flow through seal for sealing the end of the reaction vessel where the sample is received and connected to a fluid delivery system for delivering reagents to said reaction vessel through said seal, comprising the steps of: rocking said rocker apparatus to a position so that said end of said reaction vessel for receiving sample is lower than said other end; venting said other end to said waste manifold; adding a lysis buffer and proteinase K; closing said valve; rocking said rocker apparatus so that said end for receiving sample is higher than said other end; adding sample to the reaction vessel; rocking the reaction vessel back and forth with sample and lysis buffer and proteinase K therein to digest the sample to create a lysate; after digestion of the sample, rocking the reaction vessel to a position so that said other end is higher than the sample introduction end; opening said valve to vent the other end to the wast manifold; adding a phenol/chloroform based solvent to said reaction vessel; closing said valve; rocking said reaction vessel back and forth to mix said lysate and said solvent in said reaction vessel to create an emulsion of extracted amino-acids and proteins; rocking said reaction vessel to a horizontal position to increase the surface area of said emulsion and cause phase separation of said emulsion into a nucleic acid part and an organic part; educting said organic part through said valve; concentrating said nucleic acid part; and collecting said concentrated nucleic acid part.
Claim 7
Independent Claim
315 words
A method of extracting nucleic acids from a sample contained in a reaction vessel mounted on a rocker apparatus, said reaction vessel being of a flow through type having an opening on one end for receiving said sample and an opening on the other end connected to a valve for controlling eduction of fluids from said reaction vessel, said rocker apparatus having a flow through seal for sealing the end of the reaction vessel where the sample is received and connected to a fluid delivery system for delivering reagents to said reaction vessel through said seal, comprising the steps of: rocking said rocker apparatus to a position so that said end of said reaction vessel for receiving sample is lower than said other end; venting said other end to said waste manifold; adding a lysis buffer and proteinase K; closing said valve; rocking said rocker apparatus so that said end for receiving sample is higher than said other end; adding sample to the reaction vessel; rocking the reaction vessel back and forth with sample and lysis buffer and proteinase K therein to digest the sample to create a lysate; after digestion of the sample, rocking the reaction vessel to a position so that said other end is higher than the sample introduction end; opening said valve to vent the other end to the wast manifold; adding a phenol/chloroform based solvent to said reaction vessel; closing said valve; rocking said reaction vessel back and forth to mix said lysate and said solvent in said reaction vessel to create an emulsion of extracted amino-acids and proteins; rocking said reaction vessel to a horizontal position to increase the surface area of said emulsion and cause phase separation of said emulsion into a nucleic acid part and an organic part; educting said organic part through said valve; concentrating said nucleic acid part; and collecting said concentrated nucleic acid part.
References Cited (Backward Citations)
No citation records indexed.
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