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Title:
METHOD AND APPARATUS FOR FIXATION OF BIOPOLYMERS TO A SOLID SUPPORT BY CENTRIFUGATION
Document Type and Number:
WIPO Patent Application WO/1989/004874
Kind Code:
A1
Abstract:
The invention provides an apparatus and method for affixing charged biopolymers to porous supports. The invention permits standardization between separate samples and relieves the inefficiency and contamination attendant to conventional vacuum methods. The apparatus (10) includes a sample tube (12) having a plurality of sample chambers (14) with tubular exit ports (22), for receiving multiple samples, and a transverse porous support (44) at the base of exit ports (22). The support (44) is held in tight contact with the sample by a removable head (30) with exit ports (22) which effects a liquid, tight seal surrounding each exit port so as to reduce cross mixing of sample. The sample tube may be inserted into an elutant reservoir such as a centrifuge tube and centrifuged so as to effect flow of the sample through the support (44) resulting in the fixation of the biopolymer onto the support.

Inventors:
FREIER SUSAN M (US)
LONG GEORGE R (US)
Application Number:
PCT/US1988/003838
Publication Date:
June 01, 1989
Filing Date:
October 28, 1988
Export Citation:
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Assignee:
MOLECULAR BIOSYSTEMS INC (US)
International Classes:
C12Q1/68; C12Q1/6834; G01N33/543; (IPC1-7): C12Q1/68
Foreign References:
US4734363A1988-03-29
US4767699A1988-08-30
US4775619A1988-10-04
US4732847A1988-03-22
US4689294A1987-08-25
US4480040A1984-10-30
EP0152886A21985-08-28
US4722792A1988-02-02
US4550084A1985-10-29
US4522921A1985-06-11
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Claims:
original claim 8 amended; other claims
1. unchanged (1 page)] 7 A method to detect nucleic acids comprising the steps of: a) applying a fluid sample suspected of containing target nucleic acids to a porous solid support; b) centrifuging said sample and solid support to fix the nucleic acids to said support; c) contacting said fixed nucleic acids with labeled nucleic acid probe complementary to nucleic sequences in said nucleic acids fixed to said support; and d) detecting hybridized probe.
2. 8 An apparatus for affixing material contained in each of a liquid sample on a porous support by centrifugation of the liquid, said apparatus comprising: a) a tube having an axially extending chamber for holding a liquid sample, the chamber having an upper and lower ends, which are open at the upper end and are bound at the lower end by a lower floor at the bottom of the tube; b) means for positioning a porous membrane over the lower end, said means comprising a cap defining an opening, the cap being mountable on the lower end of the chamber with the orifice providing a continuing liquid passageway for the chamber, the porous support being secured between the chamber and the cap to prevent liquid flow other than through the opening.
3. 9 A multisample apparatus for affixing material contained in each of a plurality of liquid samples on separate regions of a porous support by centrifugation of the liquid, said apparatus comprising: a) a tube having a plurality of axially extending chambers for holding liquid samples, which are open at the upper end and are bound at the lower end by a lower floor at the bottom of the tube; What is claimed is: A method for the fixation of charged biopolymers in solution to a porous solid support comprising the steps of: a) applying a fluid sample suspected of containing said biopolymers to a solid support and b) centrifuging said sample and solid support so as to force said biopolymers through said solid support, whereby biopolymers in said sample are fixed to said solid support substantially irreversibly.
4. The method of Claim 1 wherein said biopolymers are nucleic acids.
5. The method of Claim 1 wherein said biopolymers are proteins.
6. The method of Claim 1 wherein said solid support is a polymer.
7. The method of Claim 3 wherein said support is selected from the group consisting of nylon, cellulose, nitrocellulose, polymerichalocarbons derivatized cellulose, or glass.
8. A method of hybridization of nucleic acids comprising the step of contacting the nucleic acids fixed to the said support obtained by the method of Claim 2 with a labelled nucleic acid probe complementary to nucleotide sequences in said nucleic acids for hybridization to said nucleic acids.
9. A method to detect nucleic acids comprising the steps of: a) applying a fluid sample suspected of containing target nucleic acids to a porous solid support; b) centrifuging said sample and solid support to fix the nucleic acids to said support; c) contacting said fixed nucleic acids with labeled nucleic acid probe complementary to nucleic sequences in said nucleic acids fixed to said support; and d) detecting hybridized probe.
10. An apparatus for affixing material contained in each of a liquid sample on a porous support by centrifugation of the liquid, said apparatus comprising: a) a tube having an axially extending chamber for holding a liquid sample, the chamber having an upper and lower ends, [which are open at the upper end and are bound at the lower end by a lower floor at the bottom of the tube] ; b) means for positioning a porous membrane over the lower end, said means comprising a cap defining an opening, the cap being mountable on the lower end of the chamber with the orifice providing a continuing liquid passageway for the chamber, the porous support being secured between the chamber and the cap to prevent liquid flow other than through the opening.
11. A multisample apparatus for affixing material contained in each of a plurality of liquid samples on separate regions of a porous support by centrifugation of the liquid, said apparatus comprising: a) a tube having a plurality of axially extending chambers for holding liquid samples, which are open at the upper end and are bound at the lower end by a lower floor at the bottom of the tube; b) means in said floor defining a plurality of ports having outwardly facing tube surface regions, where each port communicates with one of said chambers to allow liquid flow from the associated chamber out the bottom of the tube, and said tube surface regions are substantially coplanar with one another, for supporting one side of a porous support, when the apparatus is assembled for operation; c) a cap constructed for mounting against the lower floor, including means defining a plurality of openings having inwardly facing cap surface regions which are adapted to confront the corresponding outwardly facing tube surface regions so as to seal each chamber and prevent crossflow of fluids between each chamber, and to allow liquid flow from said ports through the cap in the assembled apparatus, and; d) means for attaching said cap to said tube, to hold the porous support firmly between said tube and cap surface regions.
12. The apparatus of Claim 9, wherein each port and opening in the apparatus defines a fluidflow channel, and said surface regions in at least one of said tube or cap are discrete annular regions bordering the ends of the associated channels.
13. The apparatus of Claim 10, wherein both said cap and tube include discrete annular surface regions which are substantially coextensive with another in the assembled apparatus.
14. The apparatus of Claim 9, wherein said tube is cylindrical and includes a series of radially inwardly extending members which form said chambers, and the chambers are substantially wedgeshaped in crosssection.
15. The apparatus of Claim 9, wherein said surface regions are arranged circularly around the periphery of said floor and cap, said cap has a central, axially extending opening, and said attaching means includes a bolt which can be inserted through said central opening, and a threaded central opening in said floor, for threadedly engaging said bolt, as the bolt is tightened to press the cap against the floor.
16. The apparatus of Claim 9, which further includes the porous support, which is supported between the floor and the cap, for attaching the material contained in the liquid when the apparatus is assembled.
17. The apparatus of Claim 14 wherein said porous support contains a tab which extends beyond the periphery of said floor and cap, when the apparatus is assembled.
18. The apparatus of Claim 14 for use in attaching nucleic acids in a sample, wherein said porous support is a nitrocellulose membrane.
19. The apparatus of Claim 14 wherein said surface regions are arranged circularly around the periphery of said floor and cap, said cap has a central, axially extending opening, said attaching means includes a bolt which can be inserted through said central opening and a threaded central opening in said floor, for threadedly engaging the bolt, as the bolt is tightened to press the cap against the floor, and said porous support contains a central opening through which the bolt is received.
20. The apparatus of Claim 9 which further includes a sealable reservoir for collecting liquid flowed through said porous support when the apparatus is assembled and into which the apparatus may be inserted for centrifugation.
21. The apparatus of Claim 9, which further includes an annular flange joined to the top of said tube for supporting the apparatus on the rim of said receptacle.
22. A multisample apparatus for affixing material contained in each of a plurality of liquid samples on separate regions of a porous support by centrifugation of a liquid, said apparatus comprising; a) a cylindrical tube having a plurality of axially extending, substantially wedgeshaped chambers formed from a series of radially inwardly extending members, for holding liquid samples, and which are open at the upper end, and bounded at the lower end by a lower floor at the bottom of the tube; b) a plurality of tubular members formed in the floor which communicate with said chambers and define ports arranged circularly around the periphery of said floor, and each member terminating at an annular surface region where each port communicates with one of said chambers to allow liquid flow from the associated chamber out the bottom of the tube; c) a cap constructed for mounting against lower floor, having a plurality of openings having inwardly facing cap surface regions which confront and are coextensive with the corresponding tube surface regions, and including a central, axially extending opening; d) means for attaching said cap to said tube, including a bolt which can be inserted through said central opening in said cap, and a threaded central opening in said floor, for threadedly engaging said bolt, as the bolt is tightened to press the cap against the floor to hold the porous support firmly between said tube and cap surface regions when the apparatus is assembled; and e) a porous support including a tab which extends beyond the periphery of said floor and cap, for use in attaching cellular materials in the liquid samples, when the apparatus is assembled.
23. A method for affixing material contained in liquid samples on discrete regions of a porous substrate comprising: a) placing the liquid sample into a chamber, which empties onto an associated region of a porous support; and b) centrifuging the liquid samples in the chambers to draw the liquid samples through the associated support region, thereby to fix the cellular material contained in the liquid samples onto the associated regions of the support.
24. The method of Claim 21 for use in affixing nucleic acids contained in liquid samples, wherein the porous support is a material selected from the group consisting of nitrocellulose, nylon, polymerichalocarbons, cellulose, derivatized paper and glass.
25. The method of Claim 22 further comprising the steps of removing said porous support after centrifugation to contact said support with a labeled nucleic acid probe for hybridization to said nucleic acids bound to the porous support, and detecting said labeled probe.
26. A method for affixing nucleic fluids from a plurality of liquid samples on discrete regions of a porous support comprising applying a plurality of liquid samples suspected of containing nucleic acids into the axially extending chambers of the apparatus of Claim 9 and centrifuging said samples within the apparatus.
Description:
INββRNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PC

(51) International Patent Classification 4 (11) International Publication Number: WO 89/ 0 C12Q 1/68 Al

(43) International Publication Date: 1 June 1989 (01.

(21) International Application Number: PCT/US88/03838 (81) Designated States: AT (European patent), BE ( pean patent), CH (European patent), DE (Eur

(22) International Filing Date: 28 October 1988 (28.10.88) patent), FR (European patent), GB (Europea tent), IT (European patent), JP, LU (Europea tent), NL (European patent), SE (European pat

(31) Priority Application Number : 116,403

(32) Priority Date: 3 November 1987 (03.11.87) Published

With international search report.

(33) Priority Country: US Before the expiration of the time limit for amendi claims and to be republished in the event of the r of amendments.

(71) Applicant: MOLECULAR BIOSYSTEMS, INC. [US/

US]; 10030 Barnes Canyon Road, San Diego, CA 92121 (US).

(72) Inventors: FREIER, Susan, M. ; 665 Via Barracuda,

San Clemente, CA 92672 (US). LONG, George, R. ; 18 Deer Run, Little Baltimore, Newark, DE 19711 (US).

(74) Agents: CAMPBELL, Cathryn et al.; Pretty, Schroeder, Brueggemann & Clark, 444 South Flower Street, Suite 2000, Los Angeles, CA 90071 (US).

(54) Title: METHOD AND APPARATUS FOR FIXATION OF BIOPOLYMERS TO A SOLID SUPPORT BY C TRIFUGATION

(57) Abstract

The invention provides an apparatus and method for affixing charged biopolymers to porous supports. The invention permits standar¬ dization between separate samples and relieves the inefficiency and con¬ tamination attendant to conventional vacuum methods. The apparatus (10) includes a sample tube (12) having a plurality of sample chambers (14) with tubular exit ports (22), for receiving multiple samples, and a transverse porous support (44) at the base of exit ports (22). The support (44) is held in tight contact with the sample by a removable head (30) with exit ports (22) which effects a liquid, tight seal surrounding each ex¬ it port so as to reduce cross mixing of sample. The sample tube may be inserted into an elutant reservoir such as a centrifuge tube and centri- fuged so as to effect flow of the sample through the support (44) resulting in the fixation of the biopolymer onto the support.

FOR THE PURPOSES OFLNFORMAΗON ONLY

Codes used to identify States party to thePCT on the front pages of pamphlets publishing international appli¬ cations under the PCT.

METH OD A ND APPARATUS FOR FIXATION OF BIOPOLYMERS TO A SOLID SUPPORT BY CENTRIFUGATIC#*

FIELD OF THE INVENTION

This invention relates generally to the area of immobilization of charged biopolymers and more specifically to a method using centrifugation to fix nucleic acids to a 5 porous solid support.

BACKGROUND OF THE INVENTION

Nucleic acid hybridization provides a powerful tool for diagnostic testing. Because of the specificity of binding between complementary strands of nucleic acids, labelled 0 nucleic acids, or probes, can be used to detect the presence of a specific complementary nucleotide sequence in a sample of nucleic acids.

For example, where a nucleotide sequence has been determined for a genome or portion of a genome of interest, 5 an oligonucleotide or polynucleotide probe can be construed which is complementary to a portion of that sequence. The probe is then incubated with the target nucleic acid so as to permit hybridization, after which any unhybridized probe is removed. The probe selectively hybridized to the target 0 nucleic acid can be detected by means appropriate to the label of the probe. For example, the probe may be labelled with a radioactive isotope and detected using such well- known techniques as autoradiography and scintillation counting. More recent techniques have utilized various 5 nonisotopic labels such as biotin, fluorescence or enzymes.

Typically, the target nucleic acids can be attached to a solid support prior to hybridization. Frequently the support is a membrane or filter, often nitrocellulose, nylon or a derivatized paper, although beads or particles can also be used. Application of a sample containing nucleic acids to the support is typically performed in one of several ways. Nucleic acid fragments which have been

separated, for example, by gel electrophoresis, can be eluted from the gel and applied to a membrane by a wicking or capillary process, generally referred to as a "Southern blot." Alternatively, the nucleic acids can be electroluted from the gel onto a membrane in a process known as electroblotting. In another procedure, known as colony filter, whole cells can either be grown or spotted directly on a membrane. The membrane is processed, for example by cell lysis, digestion or denaturation, to expose the nucleic acids which are then hybridized. In the dot blot method, unfractionated cellular material or partially purified nucleic acids can be applied directly to a membrane either by spotting of a small volume (1-10 μl) or by filtering of a larger volume (normally 50-200 μl) ("dot blots") using a partial vacuum to pull the liquid through the membrane.

Because the dot blot technique does not require fractionation of nucleic acids, crude clinical samples or nucleic acids isolated from clinical samples can be applied directly to the filter, thereby obviating the extensive time and labor required for electrophoresis. Moreover, plating and growth of cells is unnecessary. However, in order to obtain sufficient DNA for subsequent detection, sample volumes greater than a few microliters (μl) are generally pulled through the membrane by applying a partial vacuum to the backside of the membrane. In order to increase efficiency and standardization, more than one sample is normally processed at a time.

Various multisample vacuum manifolds are available to achieve simultaneous application of multiple samples containing nucleic acids to small "dots" or predetermined circular or elongated regions of a sheet of membrane. These manifolds typically accommodate 72 or 96 samples of about 400 μl each in separate wells. A vacuum is simultaneously applied to all of the samples and they are pulled through the membrane, depositing nucleic acids or proteins thereon. Such manifolds suffer certain drawbacks,

however. When a vacuum manifold is used for multiple samples, the force and flow rate exerted on any one sample is dependent on the flow rate at all other wells. Particularly, where some samples exhibit greater viscosity, the flow of all may be markedly decreased. As when one well becomes dry, the pressure differential changes across all samples. Moreover, the flow rate between samples in separate wells may vary greatly.

An additional drawback of available vacuum manifolds is that the membranes seal tightly only at points around the edge of the entire manifold. This design, coupled with the use of vacuum force, can lead to air leaks breaking the seals around the individual wells and the membranes with the potential for cross-mixing between samples. Furthermore, commercially available manifolds can accommodate sample sizes only as large as about 500 μl thereby reducing their usefulness for samples in which the nucleic acids or proteins may be present at a relatively low concentration. Similar requirements and difficulties surround the attachment of other biopolymers, including proteins, polysaccarides and lipids.

It is therefore apparent that there exists a need for a convenient method of fixing biopolymers, including nucleic acids, to solid supports. Such a method should ideally be readily available using systems typically present in standard laboratories, and should not require a vacuum system. Moreover, such a method should insure consistency of results among samples processed at different times. Furthermore, the system should accommodate relatively large sample volumes so as to permit analysis of samples in which biopolymers such as nucleic acids or proteins are at a low concentration. The present invention satisfies these needs and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention provides an apparatus and method for affixing charged biopolymers including nucleic acids, to porous solid supports. The invention permits standardization between separate samples and relieves the inefficiency and contamination attendant to conventional methods.

The invention provides a method for the fixation of charged biopolymers such as nucleic acids from a solution ' to a porous support by means of centrifugation. In a preferred embodiment, a fluid sample suspected of containing nucleic acids ("target" nucleic acids) is contacted with a membrane and is centrifuged so as to force the -sample through the membrane, resulting in fixation of the nucleic acids to the membrane. This process is distinguishable from filtration or precipitation aided by centrifugation where particles are merely trapped within the interstices of a filter support. Rather, the method results in the direct fixation of hybridizable nucleic acids on the porous solid support. The invention provides convenient, rapid and efficient processing of nucleic acids from samples. The samples processed may be of relatively large volume, compared to the small volumes, typically less than or equal to 0.4 ml, processed by conventional vacuum methods. The method is applicable to various charged biopolymers, including proteins, polysaccha-rides and lipids in addition to nucleic acids. For the purpose of brevity, however, it will be described principally in terms of nucleic acids.

Additionally, this invention provides an apparatus for performing centrifugal attachment of biopolymers such as nucleic acids to membranes. The apparatus includes a sample tube having a plurality of sample chambers with tubular exit ports, for receiving multiple samples and a transverse porous support at the base of the exit ports. The support is held in tight contact with the sample

chambers by a removable head with exit ports which effects a liquid-tight seal surrounding each exit port so as to reduce cross-mixing of samples. The sample tube may be inserted into an elutant reservoir such as a centrifuge tube, and centrifuged so as to effect flow of the sample through the support resulting in the fixation of nucleic acids onto the support. This apparatus permits the simultaneous processing of multiple samples which may vary significantly in volume and viscosity.

Other aspects and advantages of the present invention will become apparent from the following more detailed description of presently preferred embodiments which disclose, by way of example, the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be described in connection with the accompanying drawings in which:

Figure 1 is a side view of a centrifugation apparatus that can be used to carry out the method of the invention, about to be inserted into a centrifuge tube;

Figure 2 is a side view of the apparatus at the point of assembly, including a tube porous support and head, showing the top of the head and tube;

Figure 3 is a side view of the apparatus about to be assembled, showing the bottom of the tube, filter and head; and

Figure 4 is a cross-sectional view not to scale of the apparatus with the filter in place and head attached.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method which enables convenient and efficient fixation of charged biopolymers, such as nucleic acids or proteins, to a solid support. The method is carried out by placing a solution sample suspected of containing charged biopolymers, for example nucleic acids, in a centrifugation apparatus containing a solid support, for example a porous membrane or filter. The apparatus is then placed in a centrifuge so as to flow the sample through the membrane, resulting in nucleic acids contained in the sample being bound to the membrane. Labelled nucleic acids, or probes, can then be used to detect the presence of particular target nucleotide sequences of interest on the membrane.

As used herein, "fixation" refers to a substantially irreversible binding of a charged biopolymer, to a solid support. The binding . is due to bonding between the substance and the solid support. Fixation is not simply filtration, which refers merely to the physical trapping of a substance suspended in a liquid by passing it through a porous support. During filtration, the substance stays on the support simply because it is larger than the pores. In principle, the filtration properties of a material depend only on its pore size; the chemical composition does not matter. Filtration typically involves a biphasic system, i.e. a solid and a liquid are used, and separation is accomplished using a filter with pores sized to exclude the particles of the solid. Fixation, on the other hand, involves chemical bonding between the substance and the support; the chemical nature of the support does affect its fixation properties. Fixation can occur even if the effective molecular dimensions of the biopolymer are smaller than the pore size of the support. Using the method of this invention, nucleic acids fixed on membranes were not significantly removed by washing or by attempts to physically dislodge the material from the membrane. Moreover, fixation does not require a biphasic liquid/solid

system; as in the present invention, materials in solution can be fixed to a solid support.

Referring now to the drawings and particularly to Figures 1-4, there is shown an apparatus 10 which is preferred for use in the centrifugation method of the invention to fix cellular materials such as nucleic acids to a porous support. The apparatus may be formed from polypropylene, polystyrene or other inert sturdy material. Thus, in Figures 1 and 2, a tube 12 which is preferably cylindrical, although it may have any other appropriate shape, is provided with a plurality of axial chambers 14 for containing and separating liquid samples. The chambers may each be formed by two radially inwardly extending wall members 16 so that the chambers 14 are wedge-shaped in cross-section. Each chamber is open at the upper end 18 for introduction of the liquid samples into the chambers, and is bounded at the lower end by a planar floor 20 having exit ports 22 from each chamber for exit of the liquid samples from the chambers. As shown more clearly in Figure 3, the exit ports 22 are preferably circularly arranged around the periphery of the floor 20 and preferably have outwardly facing annular raised rings 24 joined to and surrounding each port. The rings 24 have surfaces 26 which are substantially coplanar with each other, and form a fluid flow channel shown generally at 28 in Figure 4 defining the flow of liquid from each chamber out of the tube.

The apparatus includes a removable head 30 as shown in Figures 2 and 3, for attachment to the lower end of the tube 12. The head 30 includes openings 32, preferably circularly arranged around the periphery of the head 30 so as to align with the exit ports 22 in the floor 20 of the tube 12, when the apparatus 10 is assembled. The surfaces of the openings 32 may have raised, generally annular surfaces 34 for improved sealing of the liquid flow channels between chambers. When the apparatus is assembled, the surfaces 34 surrounding each opening of the

head 30 abut the corresponding surfaces 26 of the annular rings 24 surrounding the ports 22 of the floor 20 of the tube 12. The head 30 may also include a sinuous vertical wall 36 which is useful for orientation purposes, surrounding a portion of the periphery of the head 30 and several of the openings 32, as shown in Figures 3 and 4. The wall 36, for example, may contain a slot 38 which matches a tab 40 on the outer wall of the tube 12, and may be discontinuous, as depicted in Figure 2, to provide an opening for the porous support tab 42 described further below.

In use, a porous support 44 as shown in Figures 2 and 3, is inserted between the head 30 and the tube 12 during assembly of the apparatus. The porous support may be a filter or membrane made of material suitable for attaching cellular material. For example, nitrocellulose, cellulose, derivatized paper such as ectola paper, diethylamino ethyl (DEAE) membrane, diazobenzyloxymethyl (DBM.) paper, and synthetic polymeric materials such as nylon polymerichalocarbons, and glass may be used. Nylon is preferred for use in the method of the invention to attach nucleic acids to a porous support. A preferred shape for use with the apparatus is shown in Figures 2 and 3. The porous support 44 may also be formed with a tab 42 as depicted in Figures 2 and 3 for easy insertion and removal of the support. The support may be shaped in a variety of ways, but should be sufficient in diameter to cover all of the liquid ports and openings of the tube 12 and head 30.

The head 30 is firmly attached to the tube 12, with the porous support 44 inserted between. A preferred manner of attachment includes a central opening 46 in the floor 20 of the lower end of the tube 12 as depicted in Figure 3. The opening 46 is preferably formed with threads within the opening or contains a threaded unit. The head 30 has a corresponding opening 48 and the porous support an opening 50 through which a fastening element such as a bolt 52 may be inserted during assembly to be received in the threaded

opening 46 in the tube 12. The bolt 52 may be tightened a selected amount so as to provide a liquid-tight seal.

As will be understood from the above description and the figures, when assembled, the apparatus 10 provides multiple chambers for containing liquid samples, which may be the same fluid or may each be different samples or types of fluids. A fluid flow channel 28 is defined consisting of each chamber 14, the port 22 in the floor 20 of the tube 12 in communication with the chamber 14, and the opposing opening 32 in the head 30. The arrangement of opposing surfaces of the tube ports and head openings with an interposed porous support provides discrete regions on the support for contact with the separate samples contained within each chamber. The pressure of the head against the tube created by the fastening elements, reduces the occurrence of cross flow of the samples between each chamber and leakage from the apparatus.

As illustrated in Figure 1, the apparatus 10 may also include a sealable elutant reservoir 54 for collecting waste fluid flowed through the porous support during operation. Conveniently, the reservoir 54 may be a standard centrifugation tube such as a 50 ml Falcon tube (Becton Dickinson, Lincoln Park, N.J.). For use with such a reservoir 54, the apparatus 10 preferably includes an annular flange 56 joined to the top of the tube 12 to conveniently support the apparatus on the rim 58 of the reservoir 54 when the apparatus is inserted therein.

Although the preferred apparatus for performing centrifugation is that depicted in the figures and set forth herein, other apparati known and commercially available, for example the centrifugation carrier Centrex™ (Schleicher & Schuell, Keene, NH) may be used to perform centrifugation.

To carry out the method for fixing charged biopolymers to a solid support for hybridization, liquid samples

suspected of containing a desired biopolymer material, for example a nucleic acid, are introduced into the assembled centrifugation apparatus through the top openings 18 of the tube 12. The apparatus 10 is then placed in a reservoir 54, such as a centrifuge tube, and centrifuged at a speed and for a time sufficient to effect flow of the sample through the support and into the elution reservoir 54. The speed and length of time for centrifugation will depend on the sample volume, sample viscosity and membrane porosity. Using the diagrammed device, concentrated solutions of nucleic acids (50 μg/mL) required centrifugation for about 15 minutes at 750 x g to fix the nucleic acids of the sample to a 0.45 μm Zetabind™ membrane. Larger volumes or increased viscosities require increased centrifugation times. After centrifugation, the porous support 44 is removed from the apparatus.

After the support is removed from the apparatus where the cellular material is nucleic acids, nucleic acid hybridization methods may be performed on the support to determine the presence of nucleotide sequences of interest in the attached nucleic acids. Alternatively, hybridization may be conducted within the apparatus by introducing hybridization reagent into the tube or by incubating the support with hybridization reagent and wash reagent introduced directly in the apparatus chambers or slowly centrifuged through the support. Optimum washing is achieved if the wash solution is centrifuged through the support.

Centrifugal force should be chosen so that the rate of flow of sample through the solid support yields optimum binding of target nucleic acids to the support. For example, for 0.45 μm nylon membranes, forces of 100 to 2000 x g are preferred, and preferably 750 x g.

The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the same. The examples are not intended

to limit the scope of the disclosure, or protection provided by the claims.

EXAMPLE I

The binding of target DNA to two types of nylon membranes was determined by measuring the hybridization signal of membrane bound target. Targets were prepared by incubating 1 μg of human placental DNA with 8 fmols of Hind III linearized pHSV106 plasmid (BRL, Gaithersburg, MD) in 0.3 M NaOH for 15 minutes at room temperature. Identical samples (100 μl) were applied by centrifugation (10 min at 100 x g) to Zetabind™ (CUNO, Meridian, CT) using a "microfiltration unit" (S&S, Keene, NH) and to 0.2 μm Nylon 66 in an S&S Centrex ™ unit. The membranes were prewet in 6X SSC (IX SSC is 0.15 M NaCl, 0.015 m trisodium citrate), the DNA was applied, and the membranes were rinsed with 100 μl of 2 M ammonium acetate followed by 200 μl of 6X SSC.

Membranes were removed from the units and prehybridized for 10 minutes at 60°C in 5X SSPE, 1% SDS (IX SSPE is 0.18 M NaCl, 0.01 M sodium phosphate, 0.001 M EDTA, pH7) . A synthetic DNA 22-mer complementary to the HSV-TK gene in pHSV106 was end-labeled using 32 P-ATP and T4 polynucleotide kinase to a specific activity of about 10,000 cpm/fmol. Labelled probe was purified on a c 18 Sep-Pak (Waters, Milford, MA) . Probe was added to the prehybridization solution to a final concentration of 3 nM and incubated at 60°c for 10 minutes. Membranes were washed successively 3 times for 5 minutes at 50°C in IX SSPE, 1% SDS. Membranes were counted by liquid scintillation. Results are summarized, in Table I.

TABLE I

A Comparison of Hybridization Signals on Zetabind™ and Nylon-66 Centrex™ Membranes

Membrane cpm on target cpm on control membrane 1 membrane 2

Zetabind 7523 ± 312 38 ± 3

Centrex 5967 ± 996 165 ± 25

1 1 μg human placental DNA and 8 fnol plasmid target

2 1 μg human placental DNA

EXAMPLE II

3 P-labelled plasmid DNA was used to assess the reproducibility of binding target DNA to Zetabind™ in a Hybri-Dot™ (BRL, Gaithersburg, MD) and to 0.2 μm Nylon 66 in a Centrex™ unit. Hind Ill-linearized pHSV106 plasmid was labelled using 32 P-ATP and T 4 polynucleotide kinase and purified on a P-10 spun column (Bio-Rad, Richmond, CA) . Membranes were wet in deionized water at 50°C followed by 6X SSC. Labelled DNA was mixed with human placental DNA and was applied in 0.3 M NaOH (200 μl/sample) . Samples were applied to the Centrex™ unit at 100 X g. If a sample had not passed through the membrane in 5 minutes the centrifuge speed was increased to 300 X g for 5 minutes followed by 5 minutes at 500 X g and 5 minutes at 700 X g until application was complete. The Hybri-Dot™ was used according to the manufacturer's instructions. Membranes were rinsed with 2 M ammonium acetate (100 μl) and 6X SSC

(200 μl) . Membranes were treated for 5 minutes at 60°C in

5X SSPE, 1% SDS. This treatment removed target that would be lost during hybridization. Binding of target DNA was determined by scintillation counting.

Results are shown in Table II and demonstrate increased reproducibility when centrifugation is used to fix the target. Additionally, lane 1 of the Hybri-Dot™, particularly the wells with 20 μg or more carrier DNA, filtered much more slowly than the others. This decrease was reflected in lowered binding in those wells.

A second, analogous Zetabind™ membrane was prepared in the Hybri-Dot™ and detected by autoradiography. The exposed film showed as uniform background intensity between wells reaching to adjacent, unused wells, suggesting some leakage had occurred.

TABLE II

Test of the Reproducibility of DNA Binding to Zetabind™ in a Hybri-Dot™ and to 0.2 μm Nylon 66 in a Centrex™ Unit.

Hybridot Results

Centrex Results cpm bound act carrier dot replicate 1 replicate 2 Std. Dev. (%.

0 8944 9231 2.2%

1 8006 7985 0.2%

20 5106 5752 8.4%

100 9702 9740 0.3%

300 8474 7743 6.4%

EXAMPLE III

The presently preferred apparatus was used to fix nucleic acids isolated from clinical samples on a nylon membrane and to assay them by nucleic acid hybridization for the presence of an infectious agent.

Nucleic acids were isolated from fecal specimens suspected of containing rotavirus using The Extractor™ Kit

(Molecular Biosystems, San Diego, CA) as described by the manufacturer. Briefly, 100 μl of stool was lysed and applied to The Extractor column, proteins and other interfering substances were washed away and the nucleic acids were removed from the column in 2 ml of elution reagent. To denature the viral RNA, the pH of the eluant was adjusted to pH 1.9 with HC1 and the solution was incubated for 5 minutes at room temperature. The solution was neutralized to pH 7 with NaOH. Each sample (total volume 2.15 ml) was applied to one well of the described device containing a Zetabind™ (CUNO, Meridian, CT) nylon membrane. The device was centrifuged for 20 minutes at 750 x g to force the samples through the membrane. The membrane was removed and dipped for 30 seconds in 1 M Na 2 3 followed by 30 seconds in water.

The membrane was hybridized using the SNAP R rotavirus probe kit (Molecular Biosystems, San Diego, CA) according to the manufacturer's instructions. The results for each sample were recorded as positive or negative.

The samples were also tested for rotavirus using two standard tests, electron microscopy (Brandt, et al., J. Clin. Microbiol. 13:976-981) and polyacrylamide gel electrophoresis (Herring et al., J. Clin. Microbiol. 16:473-4770) . Results of all three tests are presented in Tables III and IV.

TABLE III

Comparison of SNAP R Rotavirus System to Electron

Microscopy for the Detection of Rotavirus in Fecal

Specimens Using Centrifugation to Fix the Target

Number of Samples: EM Positive EM Negative

SNAP R Positive 41 2

SNAP R Negative 3 a 42

a These specimens showed very non-uniform distribution of rotavirus by EM.

Sensitivity = 41 = 93%

41 + 3

Specificity = 42 = 97%

42 + 2

Overall Agreement = 83 = 94% 88

TABLE IV

Comparison of SNAP R Rotavirus System using Centrifugation Device to PAGE/Silver Stained Polyacrylamide Gel Electrophoresis for the Detection of Rotavirus in Fecal Specimens

Number of Samples: PAGE Positive PAGE Negative

SNAP R Positive 111 5 SNAP R Negative 6 278

Sensitivity = 111 = 95% 111 + 6 Specificity = 278 = 98%

278 + 5

Overall Agreement = 389 = 97%

400

EXAMPLE IV

The fixation of nucleic acids to a nylon membrane by centrifugation using the preferred device was compared to filtration of nucleic acids on a nylon membrane.

Tritum-labelled E. coli DNA (17,225 cpm/μg) was used. Three and a half μg of E. coli DNA was dissolved in 3.5 ml 5 x SSC. Five hundred μl (0.5 μg, 8,612 cpm) of the DNA solution was pipetted into each of four wells of the device. Into well 1 and 2 were pipetted 0.5 ml 6 N NaOH with mixing, to denature the DNA and serve as the reference fixation samples. Into wells 3 and 4 were pipetted 1.25 ml of ethanol to precipitate the DNA, make it insoluble and serve as the filtration samples. The device was incubated for 15 minutes, then centrifuged 750 x g for 20 minutes. The filters were then cut to provide single samples which were counted by scintillation counting to measure the 3 H- DNA on the filter. Fixation samples 1 and 2 had 87% of the applied counts on the filter; filtration samples 3 and 4 had only 52% of the applied counts on the filter. Upon washing for a total of 20 minutes in 5 x SSC, the number of counts for filtration samples 3 and 4 dropped to 33% of applied counts, since ethanol-precipitated DNA on the filtration was redissolving and coming off the filter.

EXAMPLE V

The presently preferred device was used to fix proteins on a nylon membrane and to assay for activity directly on the membrane.

Calf intestinal alkaline phosphatase and glucose oxidase

(Boehringer Mannheim) were separately dissolved in 100 mM Trix HC1 buffer at concentrations of 5,000, 500, 50, 5, 0.5 and 0.05 pmol of protein. One ml of each solution was put into a well of device, and the device was centrifuged at

750 x g for 10 minutes using an aminonylon membrane (0.45 μ Zetabind ™) . The membrane was then removed, washed briefly in 10 mM Tris HCl containing 100 mM sodium chloride, 5 mM magnesium chloride and 1 mM zinc chloride. To measure enzyme activity on the membrane, the alkaline phosphatase membrane was treated with a solution containing 100 mM Tris HCl, 5 mM magnesium chloride, 1 mM zinc chloride, 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT) , incubating at 37°C for 4 hours. For development of glucose oxidase membranes, the membrane was incubated in a solution containing 100 mM imidazole HCl, pH 7.4, 100 mM sodium chloride, 1.5 μ/ml horseradish peroxidase 1.5% glucose, and 60 μ/ml o- dianidisine. Filters were then washed in 10 mM EDTA to quench. The activity of enzymes fixed to the membrane by the centrifugation was assessed by the deposition of a colored dye (blue for alkaline phosphatase, and a salmon- pink for glucose oxidase) on the membrane. Solutions as dilute as 50 pmol (7.3 ng) per ml gave detectable positive colors; controls containing no protein were colorless. While the present invention has been described in conjunction with preferred embodiments, one of ordinary skill, after reading the foregoing specification, will be able to effect various changes, substitutions of equivalents, and alterations to the compositions and methods set forth herein without departing from the spirit of the invention. It is therefore intended that the invention be limited only by the appended claims and equivalents thereof.