C - Chemistry – Metallurgy – 12 – N
Patent
C - Chemistry, Metallurgy
12
N
195/141, 23/375,
C12N 15/10 (2006.01) C07H 21/00 (2006.01) C12P 19/34 (2006.01) C12Q 1/68 (2006.01)
Patent
CA 1335963
An automated apparatus is provided implementing a new method of purifying nucleic acids from cells without the use of centrifugation. In the method, 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. The apparatus for implementing this method includes at least one extraction vessel for holding the sample and a delivery system for delivering reagents to the extraction vessel and for removing fluids therefrom. A heating system is provided for maintaining the temperature of the extraction vessel during phase separation, and a motor is used for oscillating the extraction vessel to mix fluids. The motor also rotates the extraction vessel about a horizontal axis to achieve an increase in surface area of the emulsion. The combination of the high salt concentration together with heating and increasing the surface area of the emulsion results in phase separation in 2 to 8 minutes, and thus totally eliminates the need for centrifugation. A computer system controls the extraction apparatus and a pressurized dialysis system is used to concentrate and further purify the sample after extraction.
499532
Cathcart Guy Richard
Grossman Paul David
Whiteley Norman M.
Applied Biosystems Inc.
Sim & Mcburney
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