WO2005119202A1

WO2005119202A1 - Electroelution apparatus, in particular for the extraction of macromolecules, and relative method

Info

Publication number: WO2005119202A1
Application number: PCT/IB2005/001410
Authority: WO
Inventors: Giovanni Bertoni; Faustino Vidal Aroca
Original assignee: Universita' Degli Studi Di Milano
Priority date: 2004-05-28
Filing date: 2005-05-24
Publication date: 2005-12-15
Also published as: ITMI20041082A1

Abstract

A description is given of electroelution apparatus for the extraction of biological macromolecules from samples of various origins and a method for purification of the same.

Description

ELECTROELUTION APPARATUS, IN PARTICULAR FOR THE EXTRACTION OF MACROMOLECULES. AND RELATIVE METHOD

The present invention relates to electroelution apparatus for the extraction of macromolecules of various types and origins from a sample, including a heterogeneous one, wherein they are contained, that comprises one or more test tubes placed in a thermostat-controlled container, each test tube being divided internally by a pair of membranes of different porosity which divide up the internal space, occupied by an electrolyte, into three zones: a first zone intended to contain the sample wherefrom the macromolecules have to be extracted, a second zone for collection of the eluted macromolecules and a third zone occupied solely by the electrolyte. In accordance with a preferred embodiment of the invention, in the test tube a first cylindrical body is inserted, provided on the base with a membrane permeable to the electrolyte yet able to retain the macromolecules, such as for example proteins or DNA, a second cylindrical body being inserted in said body, closed on the base by a membrane of greater porosity than the previous one and intended to hold the sample composed of a gel wherein the macromolecules are contained. By applying a difference of potential at the two ends of the test tube via electrodes integral with the same test tube, electrophoresis of the macromolecules contained in the gel takes place towards the base of the test tube, until retained by the first membrane inside the first cylindrical body. At the end of elution the second cylindrical body containing the gel is removed, and the first cylindrical body of the test tube is removed and placed upside down in a standard test tube and centrifuged to recover the eluted macromolecules. The test tubes and the cylindrical bodies with the membranes of the invention can thus be of the single-use (disposable) type. The invention also relates to a method of electroelution in which: 1) a measured quantity of electrolyte is placed in a test tube whose ends contain electrodes; 2) a first container is placed in the test tube, containing a measured quantity of electrolyte and provided on the base with a porous wall able to retain the macromolecules and allow the ions of the electrolyte to pass through; 3) a second container is placed in the first container, provided on the base with a porous wall permeable to the macromolecules, and the gel is immersed in the electrolyte in the second container; 4) finally a difference of potential is applied via the electrodes of the test tube for a predetermined time and at the end the container wherein the eluate is collected is removed and recovery is carried out by centrifuging. The extraction of macromolecules, such as for example DNA and proteins, from matrices in the gel state is a very frequent operation in molecular biology laboratories. A first method used for this operation involves breaking up the matrix chemically and later separating the macromolecules from the residues. This method however has several disadvantages, mainly because all the macromolecules frequently still contain residues of gel that may have a negative influence on the subsequent reactions that use the same macromolecule as substrate. A second method, which allows macromolecules of greater purity to be obtained, solving the problem mentioned above virtually completely, is based on the electroelution of the matrix containing the macromolecules. This allows the problems of contamination of the residues of gel to be solved yet requires however a more laborious procedure. The problem described is aggravated by the fact that there is currently no apparatus provided especially for this purpose, so that laboratory technicians use generic apparatus for electrophoresis, often designed for other purposes, which makes the procedure somewhat over-complicated. More particularly, in currently known apparatus, the eluate is collected inside the same apparatus, wherefrom it has later to be removed through operations which are not always easy and which later necessarily involve thorough cleaning and washing of the apparatus. This problem is now solved by the present invention which proposes electroelution apparatus for the extraction of macromolecules wherein the matrix containing the macromolecules is placed inside the test tubes provided at the ends of a pair of electrodes and which are inserted in a thermostat- controlled container, later connecting the electrodes to a source of supply of electrical energy. In this way the whole process of separation takes place inside the test tube which, once the eluate has been recovered, can be thrown away. Advantageously the invention involves the use of test tubes of the type commonly used in laboratories, with the addition of a few accessories which are always very low in cost. In practice, the method according to the invention allows use to be made of single-use (disposable) devices, considerably simplifying the whole process. The present invention will now be described in detail, by way of a non- limiting example, with reference to the accompanying drawings in which: • Figure 1 schematises electroelution apparatus according to the invention. • Figure 2 illustrates, sectioned, a test tube with the relative accessories for the purification of macromolecules by the method according to the invention. It should be noted that herein under reference will be made to the case of electroelution of macromolecules incorporated in a gel, but that the invention also relates to the case of macromolecules contained in other media such as for example environmental samples, biological samples of various types or samples from chemical synthesis. Referring to Figure 1, the apparatus according to the invention comprises a thermostat-controlled container denoted overall by 1, connected to devices of a known type able to maintain internally a preselected temperature which can vary within a suitable range of temperatures, and which has in the upper part a plurality of housings for the insertion of the same number of test tubes 3. Each test tube, in a plastic material, is provided, at the lid and the base, with a pair of metal electrodes whereto voltage is supplied via conductors 4 and 5. The test tubes which are placed in the container 1 are illustrated in detail in Figure 2. 2 here denotes the body of the test tube provided with lid 14 and two electrodes 17 and 10, wherein a hollow cylindrical body 6 is placed, closed at the base by a membrane 7 and, at the top, by the lid 14 of the body of the test tube. A series of limit bracket elements 8 project from the internal part of the test tube and serve to prevent the cylindrical body 6, when it is inserted fully in the test tube, from reaching as far as the base of the same, instead it stops at a certain distance, in such a way that a certain space remains between the membrane 7 and the base of the test tube that defines a chamber 9 for depositing of the electrolyte, as will be explained in greater detail herein below. In the cylindrical body 6 a second cylindrical container 11 is placed, closed on the base by a membrane 12. The cylindrical container 11 is shorter than the container 6, in such a way that, in the space between the two membranes, a chamber 13 is defined wherein the eluate is collected. The interior of the cylinder 11 , which is closed above by the lid 14, defines a chamber 15 wherein the gel is placed, for example in the form of a tablet 16 or the like, wherefrom the macromolecules are extracted. The chamber 9 and 13 and the space of the chamber 15 not occupied by the gel contain the electrolyte. The electrode 17 attached to the lid is in electrical contact with the lower electrode 10, via the electrolyte. The membrane 7 has such porosity as to allow the passage of the electrolyte yet retain the macromolecules to be eluted from the gel. The porosity of the membrane 7 will preferably be from 0.045 μm to 0.015 μm and preferably 0.025 μm approximately. The membrane 12 which closes the base of the chamber 15 instead has greater porosity, for example from 0.3 to 0.6 μm, and preferably 0.45 μm approximately, so as to allow the free passage of the macromolecules and of the electrolyte yet prevent any particles of gel from passing into the chamber

13 for collection of the eluate. Membranes of this type, 7 and 12, are available commercially, produced by the firm Millipore. The test tube is prepared for use by inserting the cylindrical body 6 in the body 2 of the test tube, having previously placed on the base of the body 2 a volume of electrolytic solution equal to the volume of the chamber 9, and the cylindrical body 11 is then inserted in the body 6, having previously placed in the body 6 a volume of electrolytic solution equal to the volume of the chamber 13; a tablet of gel to be eluted is placed in the chamber 15 of the cylindrical body 11 and the space of the chamber 15 not occupied by the gel is filled with the electrolyte solution, closing with the lid 14. At this point the test tube is ready and is placed in the thermostat- controlled container 1 (Fig. 1) of the electroelution apparatus, provided with a base and lid. Here the electrode 10 comes into contact with a plate of a conductor 5 placed in the base of the apparatus. Once the apparatus is closed, the upper electrode 17 also comes into contact with a similar plate 4 placed in the lid of the apparatus. The plates 4 and 5 are in turn connected to a source of supply of electrical energy with appropriate voltage. In order to contrast the effect of heating due to the passage of current between the electrodes, the internal temperature of the container 1 is regulated in order to maintain it at the value specified for the type of material to be handled, and the procedure is started by supplying electrical current through the conductors 4 and 5. The difference of potential which is applied between the electrodes causes the migration of the macromolecules contained in the gel through the membrane 12, transported by the current via the electrophoretic buffer with which the test tube is filled. The pores in the membrane 12 are such as to retain the gel yet allow the free passage of the molecules. When the latter reach the base of the cylindrical body 6 they are retained by the membrane 7 which allows only the electrolyte to pass through. The macromolecules are therefore collected on the base of the chamber 13, against the membrane 7. At the end of the procedure the test tubes are removed from the thermostat-controlled apparatus 1 , the cylindrical body 1 1 is withdrawn and thrown away, the cylindrical body 6 which contains the macromolecules is removed and retained, and the body of the test tube 2 is thrown away. The cylindrical body 6 is then inserted, upside down, in a fresh standard test tube and centrifuged briefly to collect the eluted product. A person skilled in the art may later foresee various modifications and variants, which must however be considered as all comprised within the scope of the present invention.

Claims

1. Electroelution apparatus for the extraction of macromolecules from samples of various origins, characterised in that it comprises a test tube divided internally by a porous membrane into a first chamber containing an electrode, intended to receive the electrolyte, and a second chamber intended to collect the eluate, said second chamber being, in turn, divided by a second porous membrane into a first zone for collecting the eluate and a second zone intended to contain the sample, a second electrode being provided in said second zone.

2. Electroelution apparatus according to claim 1, characterised in that it comprises: • a test tube provided with an electrode at the base; • a cylindrical container, closed on the base by a first membrane designed to retain the molecules to be eluted and to allow the electrolyte to pass through, said container being inserted in said test tube and reaching a certain distance from the base of the same; • a second cylindrical container designed to contain the sample to be eluted, inserted in said cylindrical container and closed on the base by a second membrane porous to macromolecules and able to retain the material whereof the sample is composed; • a second electrode inserted in said second cylindrical container.

3. Electroelution apparatus according to claim 2, characterised in that said first membrane has porosity from approximately 0.045 μm to approximately 0.015 μm and said second membrane has porosity from approximately 0.30 μm to approximately 0.60 μm.

4. Electroelution apparatus according to claim 3, characterised in that said first membrane has porosity of approximately 0.025 μm and said second membrane has porosity of approximately 0.45 μm.

5. Electroelution apparatus according to any one of the previous claims, characterised in that one electrode is through and integral with the base of the test tube and a second electrode is inserted and integral with the closure plug of said test tube and dips into said second cylindrical container.

6. Electroelution apparatus according to any one of the previous claims, characterised in that it provides a thermostat-controlled container provided with housings for one or more test tubes and comprising, on the base wall and on the upper one, means suitable for supplying electrical current to pairs of electrodes provided in the test tubes.

7. Method of elution for the purification of macromolecules, characterised in that the following phases are foreseen: • a measured quantity of electrolyte is placed in a test tube whose ends contain electrodes; • a first container is placed in the test tube, containing a measured quantity of electrolyte, provided on the base with a porous wall able to retain the macromolecules and allow the ions of the electrolyte to pass through; • a second container is placed in the first container, provided on the base with a porous wall permeable to the macromolecules; • in the second container the gel is immersed in the electrolyte; • finally a difference of potential is applied via the electrodes of the test tube for a predetermined time, and • at the end the container wherein the eluate is collected is removed and recovery through centrifuging is carried out.

8. Electroelution method and apparatus in particular for the extraction from a gel of macromolecules, such as for example DNA and proteins, as described and illustrated.

9. Electroelution method and apparatus according to any one of the previous claims for the extraction of macromolecules contained in complex samples, different from gelled matrices used in the laboratory, such as for example environmental samples, biological samples of various types and origins and samples from chemical synthesis.