Patent US2099480A successfully refreshed from Google Patents.
Patent EP0215533A2 successfully refreshed from Google Patents.
EP0215533A2
Expired / Lapsed
EP
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A method of extracting nucleic acids from cells
Assignee: Applied Biosystems Inc
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Filing: 1986-01-07
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Granted: —
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Expires: 2006-01-07
Abstract
Abstract A method of extracting nudeic adds from cells without the use of centrifugation in which cells are treated with proteinase K in the presence of a lysis buffer having a high concentration of salt. The lysate is mixed with a phenol-based solvent system, thereby creating an emulsion which is heated to a temperature of at least 35 °C to promote phase separation. The rate of phase separation is also enhanced by increasing the surface area of the emulsion. 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 the remaining nucleic acid solution.
Inventors
- 👤 Guy Richard Cathcart
- 👤 Paul David Grossman
- 👤 Norman Whiteley
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-05 10:06
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/1003
Claims Analysis
Total Claims: 29
Claim 1
Independent Claim
104 words
A method of extracting nucleic acids from cells which includes the steps of (a) creating a lysate by treating said cells with proteinase K in the presence of a lysis buffer; (b) mixing said lysate with a phenol-based solvent system, thereby creating an emulsion; and (c) separating the phases into an aqueous phase containing said nucleic acids and an organic phase containing phenol and denatured proteins characterised in that (i) a high concentration of salt is provided in said lysis buffer (ii) said emulsion is heated prior to separation to a temperature greater than 35°C and (iii) said organic phase is removed without centrifugation.
Claim 2
Independent Claim
104 words
A method of extracting nucleic acids from cells which includes the steps of (a) creating a lysate by treating said cells with proteinase K in the presence of a lysis buffer; (b) mixing said lysate with a phenol-based solvent system, thereby creating an emulsion; and (c) separating the phases into an aqueous phase containing said nucleic acids and an organic phase containing phenol and denatured proteins characterised in that (i) a high concentration of salt is provided in said lysis buffer (ii) said emulsion is heated prior to separation to a temperature greater than 35°C and (iii) said organic phase is removed without centrifugation.
Claim 3
Dependent Claim
14 words
A method according to claim 1 wherein the said temperature is greater than 45°C.
Claim 4
Dependent Claim
13 words
A method according to claim 1 wherein the said temperature is about 55°C.
Claim 5
Independent Claim
29 words
A method according to any preceding claim wherein after the heating step but prior to separation, the surface area of the emulsion is increased thereby further promoting phase separation.
Claim 6
Independent Claim
36 words
A method according to any preceding claim wherein the steps of mixing, heating (surface extension if employed) and separating are repeated, in order, N times, where N is a number greater than or equal to 1.
Claim 7
Independent Claim
40 words
A method according to any preceding claim wherein after final separation the remaining aqueous phase is mixed with chloroform thereby creating an emulsion and the heating and separation steps are repeated to leave an aqueous phase containing said nucleic acids.
Claim 8
Dependent Claim
65 words
A method according to claim 6 as dependent on claim 4 and claim 5 wherein after the steps of mixing, heating, surface extension and separation have been repeated N times, the remaining aqueous phase is mixed with chloroform thereby creating an emulsion, and the steps of heating, surface extension and separation are repeated in that order to leave an aqueous phase containing said nucleic acids.
Claim 9
Dependent Claim
16 words
The method of claim 5 wherein the remaining aqueous phase after mixing with chloroform is dialyzed.
Claim 10
Independent Claim
21 words
A method according to any preceding claim wherein said phenol-based solvent system comprises a mixture of phenol, chloroform, and an antisurfactant.
Claim 11
Dependent Claim
18 words
The method of claim 8 wherein the ratios of phenol to chloroform to isoamyl alcohol are about 50:48:1.
Claim 12
Independent Claim
13 words
The method of any preceding claim wherein said lysis buffer includes a detergent.
Claim 13
Dependent Claim
23 words
The method of claim 10 wherein said lysis buffer inlcudes one or more of a chaotropic agent, a chelating agent, and a buffer.
Claim 14
Independent Claim
22 words
A method according to any preceding claim wherein the molar concentration of salt in said lysis buffer is from 0.5 and 2.
Claim 15
Dependent Claim
18 words
A method according to claim 13 wherein said lysis buffer comprises urea, NaCl, a detergent, Tris-HCℓ, and EDTA.
Claim 16
Dependent Claim
23 words
The method of claim 14 wherein the lysis buffer comprises 8M urea, 1M NaCl, 1% SDS, 50mM Tris-HCl, and 10mM EDTA, pH 8.0.
Claim 17
Dependent Claim
165 words
Apparatus for extracting nucleic acids from cells comprising: extraction vessel means (11-15) for holding said cells: delivery means (100) for delivering reagents to said extraction vessel means, and for removing fluids from said extraction vessel means: heater means (221-225) for heating said extraction vessel means; temperature measuring means (231-235) for measuring the temperature of said extraction vessel means; motor means (200) for oscillating said extraction vessel means to mix said cells and said reagents; computer means (400) for controlling said heater means, said motor means and said delivery means, for controlling volume of flow of said reagents into said extraction vessel means, for monitoring the temperature measured by said temperature measuring means, for monitoring flow of fluids from said extraction vessel means, for timing the heating of said extraction vessels by said heating means, and for timing the mixing by said motor means, said computer means operating according to a preselected instruction set to cause extraction of said nucleic acids from said cells without centrifugation.
Claim 18
Dependent Claim
165 words
Apparatus for extracting nucleic acids from cells comprising: extraction vessel means (11-15) for holding said cells: delivery means (100) for delivering reagents to said extraction vessel means, and for removing fluids from said extraction vessel means: heater means (221-225) for heating said extraction vessel means; temperature measuring means (231-235) for measuring the temperature of said extraction vessel means; motor means (200) for oscillating said extraction vessel means to mix said cells and said reagents; computer means (400) for controlling said heater means, said motor means and said delivery means, for controlling volume of flow of said reagents into said extraction vessel means, for monitoring the temperature measured by said temperature measuring means, for monitoring flow of fluids from said extraction vessel means, for timing the heating of said extraction vessels by said heating means, and for timing the mixing by said motor means, said computer means operating according to a preselected instruction set to cause extraction of said nucleic acids from said cells without centrifugation.
Claim 19
Dependent Claim
87 words
Apparatus according to claim 16 wherein said extraction vessel means is configured such that when it is rotated about a horizontal axis by an angle, a fluid contained therein will have a larger surface area than when said extraction vessel means is not rotated, and wherein said motor means is arranged for rotating said extraction vessel means to said angle and for holding said extraction vessel means at said angle at least for a period to permit phase separations of emulsions contained in said extraction vessel means.
Claim 20
Dependent Claim
17 words
Apparatus according to claim 16 or 17 wherein said extraction vessel means comprises a plurality of tubes.
Claim 21
Dependent Claim
39 words
Apparatus according to any of claims 16 to 18 further comprising dialysis means (300) coupled to said extraction vessel means via said delivery means, and said delivery means is arranged for delivering fluids to and from said dialysis means.
Claim 22
Dependent Claim
19 words
Apparatus according to claim 19 wherein said dialysis means comprises recirculation means for recirculating and filtering dyalysate during dialysis.
Claim 23
Independent Claim
105 words
A device for enhancing the rate of phase separation of an emulsion made up of an aqueous phase and an organic phase, comprising: an extraction vessel (11-15), said extraction vessel having a cross-section area A1 in a horizontal plane through said extraction vessel, and having a cross-sectional area A2 in a plane oriented at an angle 8 relative to the horizontal, where A2 is greater than A1; motor means having a driveshaft coupled to said extraction vessel for rotating said extraction vessel by angle θ, and for holding said extraction vessel at said angle θ for a period during or prior to said phase separation.
Claim 24
Independent Claim
105 words
A device for enhancing the rate of phase separation of an emulsion made up of an aqueous phase and an organic phase, comprising: an extraction vessel (11-15), said extraction vessel having a cross-section area A1 in a horizontal plane through said extraction vessel, and having a cross-sectional area A2 in a plane oriented at an angle 8 relative to the horizontal, where A2 is greater than A1; motor means having a driveshaft coupled to said extraction vessel for rotating said extraction vessel by angle θ, and for holding said extraction vessel at said angle θ for a period during or prior to said phase separation.
Claim 25
Dependent Claim
13 words
A device as in claim 21 including means for heating said extraction vessel.
Claim 26
Dependent Claim
31 words
A device according to claim 21 including means for withdrawing the lower phase and control means for said withdrawing means, said control means including means for detecting a change in conductivity.
Claim 27
Independent Claim
197 words
A device for the automated control of reactions involving a phase separation comprising: holding means for holding reagents to be reacted; computer means; one or more extraction vessels (11-15); first valve block means having a plurality of ports, said first valve block means being responsive to signals from said computer means for directing delivery of said reagents to said extraction vessel(s); heating means responsive to signals from said computer means for heating said extraction vessel(s); delivery means arranged in response to said computer means for delivering said reagents to said first valve block means from said holding means, and from said first valve block means to said extraction vessel(s), and for removing fluids from bottom portion(s) of said extraction vessel(s), and for delivering said fluids from said bottom portion(s) to said first valve block means: motor means responsive to signals from said computer means for rocking said extraction vessel(s) to mix reagents contained therein; measuring means coupled to said first valve block means for receiving fluids therefrom and for measuring conductivity of said fluids received from said first valve block means, and providing a signal to said computer means when said conductivity is above a preselected threshold.
Claim 28
Independent Claim
197 words
A device for the automated control of reactions involving a phase separation comprising: holding means for holding reagents to be reacted; computer means; one or more extraction vessels (11-15); first valve block means having a plurality of ports, said first valve block means being responsive to signals from said computer means for directing delivery of said reagents to said extraction vessel(s); heating means responsive to signals from said computer means for heating said extraction vessel(s); delivery means arranged in response to said computer means for delivering said reagents to said first valve block means from said holding means, and from said first valve block means to said extraction vessel(s), and for removing fluids from bottom portion(s) of said extraction vessel(s), and for delivering said fluids from said bottom portion(s) to said first valve block means: motor means responsive to signals from said computer means for rocking said extraction vessel(s) to mix reagents contained therein; measuring means coupled to said first valve block means for receiving fluids therefrom and for measuring conductivity of said fluids received from said first valve block means, and providing a signal to said computer means when said conductivity is above a preselected threshold.
Claim 29
Independent Claim
32 words
A device according to any of claims 21 to 24 further comprising dialysis means coupled to receive fluids from said extraction vessel, and for dialysing said fluids received from said extraction vessel.
References Cited (Backward Citations)
No citation records indexed.
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