Cellular component extraction process in a disposable filtration vessel

• US 5,114,858 A
• Filed: 06/26/1990
• Issued: 05/19/1992
• Est. Priority Date: 06/26/1990
• Status: Expired due to Term

• Current Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Original Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Inventors: Rosanio, Louis, Williams, John G.
• Primary Examiner(s): Hill, Jr., Robert J.
• Assistant Examiner(s): Chan, William
• Application Number:
  US07/543,928
• Time in Patent Office: 693 Days
• Field of Search: 935/19, 935/20, 935/21, 435/681, 435/267, 435/259, 435/270, 435/6, 435/287, 435/311, 436/177, 422/101, 538/28, 538/72
• US Class Current: 435/270
• CPC Class Codes: C12M 47/06 Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material lysis of microorganisms C12N1/06; extracting or separating nucleic acids from biological samples C12N15/1003
• CPC Class Codes: C12N 1/08 Reducing the nucleic acid content
• CPC Class Codes: C12N 15/1017 by filtration, e.g. using filters, frits, membranes
• CPC Class Codes: Y10T 436/25375 Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]

Abstract

A method of extracting cellular components, e.g., nucleic acids, particularly DNA, from biological, expecially solid tissue, samples, particularly from plants is provided, whereby all of the steps can be performed in one vessel, and the entire process can be automated. A vessel suitable for this process is provided, as well as a system for the automated performance of the process steps.

Claims

1. A method of separating nucleic acids from a biological sample, comprising:

(a) releasing the intracellular contents from the sample in the extraction vessel by operating the disrupting means;

b) separating aqueous solvent-soluble components of the cells into one phase and organic solvent-soluble components of the cells into a second phase in a first chamber of an extraction device comprising;

(i) the first chamber and a second chamber separated by a radially extending wall with means for allowing fluid to pass through, the first chamber and the second chamber having openable ends;

(ii) a plunger means for insertion into said first chamber and a means for retaining the plunger means in the first chamber to retain the sample between inserted end of the plunger means and the radially extending wall;

(iii) a means associated with the plunger means and the radially extending wall for disrupting the sample, whereby nucleic acids are released therefrom;

(iv) a means for sealing between the plunger means and the first chamber to block fluid communication of the released nucleic caids through the openable end of the first chamber;

c) filtering the aqueous phase sample into a tube means having a barrel, the tube means being insertable in the second chamber for accumulating nucleic acid-containing fluid introduced therein from the first chamber, the tube means further including a filter means having a first and second side such that the aqueous solvent phase is on the second side of the filter means and the organic solvent phase and any cellular debris is on the first side of the filter means upon transfer of the nucleic acid-containing fluid from the the second chamber to the barrel of the tube means, and a means for sealing between the tube means and the second chamber to block fluid communication of the released nucleic acids through the openable end of the second chamber;

d) precipitating nucleic acids out of the aqueous phase in the barrel of the tube means; and

e) filtering the precipitated nucleic acids onto the second side of said filter means.

2. A method of claim 1, wherein the biological sample is a solid tissue sample.

3. A method of automatically separating nucleic acids from a biological sample by using an apparatus comprising:

(a) a plurality of extraction vessels for storing, transporting and processing biological samples therein, each extraction vessel comprising;

(i) a lower chamber and an upper chamber separated by a radially extending wall with means for allowing fluid to pass through, the lower chamber and the upper chamber having openable ends;

(ii) a tube means having a barrel, the tube means insertable into the upper chamber for accumulating nucleic acid-containing fluid introduced therein from the lower chamber, the tube means further including a filter means having a first and second side;

(iii) plunger means for insertion into the lower chamber;

(iv) means for retaining the plunger means in the lower chamber to retain the sample between the inserted end of the plunger means and the radially extending wall;

(v) disrupting means associated with the plunger means and the radially extending wall for disrupting the sample upon relative movement between the extraction vessel and the sample, whereby nucleic acids are released therefrom; and

(vi) means for sealing between the plunger means and the first chamber to block fluid communication of the release nucleic acids through the operable end of the first chamber;

(b) a turntable having port stations with a first array of ports circumferentially distributed therearound for receiving the extraction vessels without the filter tubes inserted therein and a second array of ports distributed circumferentially therearound in radial alignment with the first ports for subsequently receiving the filter tubes, said turntable having more port stations thereon for insertion of extraction vessels and filter tubes than processing stations to provide for extended treatment times as the turntable indexes;

(c) means for inserting both the extraction vessels and filter tubes into the turntable and means for removing both the extraction vessels and filter tubes from the turntable after separating the nucleic acid material extracted from the biological sample; and

(d) processing means disposed at stations adjacent the turntable for processing the sample to separate the nucleic acid material from the sample, wherein the processing means comprises;

(i) means for introducing treating fluids into the extraction vessels and filter tubes;

(ii) means for mixing and treating fluids with the samples;

(iii) means for transferring fluid between the extraction vessels and filter tubes to effect treating, washing, and filtering of the samples and the nucleic acids therein;

(iv) means for identifying each sample, and

(v) means for transferring nucleic acids filtered from the treating fluids to storage means for subsequent analysis, said storage means for subsequent analysis comprising an array of microtiter dishes on an indexable table located below the turntable,

said method comprising;

(a) releasing the intracellular contents form the sample in the extraction vessel by operating the disrupting means;

(b) separating aqueous solvent-soluble components of the sample into a first phase and organic solvent-soluble components of the sample into a second phase in the lower chamber of the extraction vessel;

(c) filtering the aqueous phase into the tube means, accumulating the nucleic acid-containing fluid introduced therein rom the lower chamber such that the aqueous phase is on the second side of the filter means and the organic phase and any cellular debris is on the first side of the filter means upon transfer of the nucleic acid-containing fluid from the upper chamber to the barrel of the tube means, at the same time blocking fluid communication of the release nucleic acids through the openable end of the upper chamber by means of the sealing means;

(d) precipitating the nucleic acids out of the aqueous phase in the barrel of the tube means; and

(e) filtering the precipitated nucleic acids onto the second side of the filter means.