HK1014959A

HK1014959A - Method and apparatus for dna extraction

Info

Publication number: HK1014959A
Application number: HK98119192.9A
Authority: HK
Inventors: 蒂莫西‧马丁‧伊文斯; 罗伯特‧体‧唐
Original assignee: 普罗金工业有限公司
Priority date: 1995-06-08
Filing date: 1996-06-11
Publication date: 1999-10-08

Description

Method and apparatus for extracting DNA

The present invention relates to a method and apparatus for extracting DNA from a cell suspension. In particular, the present invention relates to a method and apparatus for extracting plasmid DNA from microorganisms and genomic DNA from microorganisms and animal cells.

Background

The use of recombinant DNA techniques generally involves the use of plasmids or cosmids in the cloning or manipulation of target DNA. Generally, plasmids or cosmids are amplified by culturing microorganisms carrying the plasmid or cosmid and finally extracting the plasmid or cosmid from the culture. The extracted DNA often needs to be purified.

Automated or semi-automated procedures have been developed for certain recombinant DNA techniques, particularly DNA purification and sequencing, and many methods still require manual manipulation. This is particularly the case for methods of plasmid DNA extraction. Conventional methods such as alkaline lysis or boiling lysis (see, Sambrook et al, molecular cloning: A laboratory Manual, second edition, Cold spring harbor laboratory Press, Cold spring harbor, New York, 1989) involve a number of steps. Whether it is successful depends on the level of skill of the researcher. Therefore, repeated application of the method by unskilled experimenters can lead to differences in the results produced.

It is often the case that plasmid or cosmid DNA must be extracted from many different cultures. Since microtiter plates are used in some recombinant DNA techniques, extraction of DNA from hundreds of different cultures is required in this case. Conventional extraction procedures are not suitable for automatic, or even semi-automatic, operation. Therefore, the extraction of plasmid or cosmid DNA from a large number of individual cultures is a time-consuming and labor-intensive operation.

There is therefore a need for a method for extracting plasmid or cosmid DNA from microbial cultures that is simpler and more reproducible than existing procedures. There is also a need for an extraction method that is adapted to be automated so that large quantities of culture can be processed.

Likewise, applications of recombinant DNA techniques and DNA analysis techniques involve the use of genomic DNA from animal blood and other body fluids. Conventional methods such as extraction with organic solvents (see, Sambrook et al, molecular cloning: A laboratory Manual, second edition, Cold spring harbor laboratory Press, Cold spring harbor, New York, 1989) involve a number of steps, depending on the level of skill of the researcher. Therefore, repeated application of the method by unskilled experimenters can lead to differences in the results produced.

It is often the case that genomic DNA must be extracted from a large number of samples, particularly for the use of DNA in polymerase chain reactions and DNA sequencing. Since microplates are used in some recombinant DNA techniques and genetic analysis, DNA extraction from hundreds of samples is required in this case.

There is therefore a need for a method for extracting genomic DNA from blood and other body fluids that is simpler and more reproducible than existing procedures. There is also a need for an extraction method that is adapted to be automated so that a large number of samples can be processed.

Summary of The Invention

It is an object of the present invention to provide a method for extracting DNA from a cell suspension which overcomes the disadvantages of the prior art extraction methods. In particular, it is an object of the present invention to provide a method for selectively extracting plasmid DNA from a cell suspension, which overcomes the disadvantages of the existing methods for extracting plasmid DNA.

It is another object of the present invention to provide a device for extracting DNA from a cell suspension.

The term "cell suspension" as used above and hereinafter includes cultures of animal cells, cultures of microorganisms, body fluids such as blood, lymph, urine and semen and other dispersions of individual cells in a matrix of body fluids.

The term "plasmid" as used above and hereinafter refers to DNA molecules referred to as plasmids and cosmids.

According to a first embodiment of the present invention, there is provided a method for extracting DNA from a cell suspension, the method comprising the steps of:

1) adding the cell suspension to a filtration device having a hollow membrane filter;

2) if necessary, filtering to remove the medium suspending the cells;

3) adding a lysis solution to said cells and incubating said cells for a sufficient time to release DNA; and

4) the lysis solution containing the DNA was filtered.

According to a second embodiment of the present invention, there is provided a method for extracting DNA from a cell suspension, the method comprising the steps of:

2) if necessary, filtering to remove the medium suspending the cells;

4) filtering the lysis solution containing the DNA;

5) adding the filtrate of step (4) to an ion exchange medium;

6) washing the ion exchange medium with a first solution to elute substances other than DNA; and

7) washing the ion exchange medium with a second solution to elute the DNA.

According to a third embodiment of the present invention, there is provided a method for extracting plasmid DNA from a culture of microorganisms, the method comprising the steps of:

1) adding a culture of the plasmid DNA-carrying microorganism to a filtration apparatus having a hollow membrane filter;

2) if necessary, the medium is removed by filtration;

3) adding a lysis solution to the microorganism and incubating the microorganism for a sufficient time to release plasmid DNA therefrom; and

4) the lysis solution containing the plasmid DNA was filtered.

According to a fourth embodiment of the present invention, there is provided a method for extracting plasmid DNA from a culture of microorganisms, the method comprising the steps of:

2) if necessary, the medium is removed by filtration;

3) adding a lysis solution to said microorganism and incubating said microorganism for a sufficient time to release plasmid DNA; and

4) filtering the lysis solution containing the plasmid DNA;

5) adding the filtrate of step (4) to an ion exchange medium;

6) washing the ion exchange medium with a first solution to elute substances other than plasmid DNA; and

7) washing the ion exchange medium with a second solution to elute the plasmid DNA.

According to a fifth embodiment of the present invention, there is provided an apparatus for culturing cells and extracting DNA, the apparatus comprising:

a container having an open end and a closed end, the closed end comprising a hollow membrane filter allowing fluid communication between the interior and exterior of the container; and

a movable seal sealing said closed end.

According to a sixth embodiment of the present invention, there is provided an apparatus for culturing cells and extracting DNA, comprising a plurality of the apparatuses according to the fifth embodiment.

According to a seventh embodiment of the present invention, there is provided a kit for culturing cells and extracting DNA, comprising at least one device according to the fifth embodiment or at least one device according to the sixth embodiment.

The term "hollow membrane filter" is used interchangeably with the term "hollow fiber filter" in the art. However, only the former term is used herein.

Referring to the first embodiment of the present invention, it can be appreciated that the principle of the method is to lyse the relevant cells and separate a lysate containing DNA from the cell debris using a hollow membrane filter. Genomic DNA can be extracted from animal cells and microorganisms and cultures of these cells using this method. This method can also be used to extract plasmid DNA from microorganisms, as described in detail below. However, when used to extract plasmid DNA, milder lysis conditions were used. This method has a great advantage over known DNA extraction procedures, where the entire procedure is performed in a hollow membrane filtration unit.

For the first step of the method according to the first embodiment, any cells that are adapted to the lysis of step (3) of the method can be used for the entire procedure. If they are first dispersed to form a suspension, the aggregated cells can also be used for this procedure. Methods for forming aggregated cells into a dispersion are known to those skilled in the art. As described above, the cell suspension may be a suspension of cultured cells. The culture can be prepared using conventional methods and conditions. The culture used in the method of the invention may be an additionally produced culture sample or, as described in detail below, may be produced in a device comprising a hollow membrane filter. Thus, in the latter case, the culture is prepared in situ.

Excess suspension medium can be filtered off, leaving concentrated cells on the hollow membrane filter (step (2) of the method). Filtration can be achieved by applying a positive pressure on the inlet side of the membrane filter or a negative pressure on the outlet side of the membrane filter. Positive pressure is typically applied by centrifugation or by using air pressure. Typically, the negative pressure may be provided by drawing a vacuum, which is the preferred method of filtration. The previous filtration method is also applicable to the subsequent steps of the first embodiment, as well as to all the steps of the second embodiment.

The method of the first embodiment further comprises the step (2a) of washing the cells collected on the vacuum membrane filter of step (2). Suitable wash solutions for step (2a) are also known to those skilled in the art and typically comprise a buffer solution of sucrose or glucose. Preferred wash solutions include:

16.5% sucrose

36mM Tris.HCl(pH8.0)

55mM EDTA

The wash solution also includes an enzyme that breaks up cell wall components. The enzyme is typically lysozyme.

Washing of the cells can be accomplished by passing a volume of wash solution through the membrane filter under positive or negative pressure. The volume of wash solution used is not critical and most conveniently is about the same as the initial volume of cell suspension.

The lysis solution of step (3) of the first embodiment is preferably a buffer containing a chaotropic agent with or without a detergent. Suitable chaotropic agents include guanidine hydrochloride, sodium iodide, sodium perchlorate and salts of guanidines such as, for example, guanidine thiocyanate. Typically the chaotropic agent is present at a concentration of 3-6M. Even a saturated solution of chaotropic agent (in some cases about 8M) may be used. Suitable detergents include Tween^TM20，TritonX-100^TM，Nonidet^TM P-40，Brij58^TMSodium deoxycholate, N-lauroylsarcosine, and the like. Typically the detergent is present at a concentration of 0.05-5%. For example TritonX-100^TMMay be present in the lysis solution at a concentration of 0.75-5%. The pH of the lysis solution is usually 5-9. The lysis solution may also contain a denaturing agent such as urea.

Preferred lysis solutions include:

4M guanidine thiocyanate

0.1M sodium acetate (pH5.0)

5％TritonX-100^TM

3M Urea the lysis solution is particularly suitable for extracting genomic DNA from animal cells.

It is preferred to use a minimal volume of lysis solution to avoid dilution of the extracted DNA. Typically, a volume of lysis solution is used that is about the culture volume.

The cells are incubated in the presence of the lysis solution for a sufficient period of time to release large amounts of genomic DNA. For animal cells, incubation times of 3-15 minutes are suitable.

The lysis solution containing the extracted DNA is filtered in the final step of the first embodiment of the present invention. The DNA may be concentrated or transferred to another solution using techniques known to those skilled in the art. For example, the use of organic solution can be precipitated DNA or plasmid DNA solution gel filtration or dialysis.

Steps (3) and (4) of the method of the first embodiment are repeated one or more times to increase the yield of DNA. The repeated steps involve simply adding fresh lysis solution, incubating for a certain time to release additional DNA, and filtering the lysis solution.

The method of the second embodiment provides a conventional method for obtaining purified DNA. The first four steps of the process may be carried out as described above for the first embodiment, including optional step (2 a). Ion exchange media suitable for use in step (5) of the process are known to those skilled in the art and include inert substrates substituted with known groups such as DEAE (diethylaminoethyl), QAE (quaternary aminoethyl) and Q (quaternary ammonium). A preferred ion exchange medium is silica.

The lysis solution of step (3) is designed to allow the DNA to bind to the ion exchange medium, eluting the other compounds contained in the lysate. For the combination with a silica filter, a particularly preferred lysis solution is the lysis solution defined in step (3) of the above-described first embodiment.

Washing the ion exchange media typically involves passing portions of the first solution through the media. The first solution is preferably a solution that does not dissolve DNA. For use in combination with silica ion exchange media, an 80% isopropanol solution in water is preferred.

Elution of DNA at step 8 of the method may be accomplished by passing a solution that dissolves DNA through the medium. Typical solutions include water containing low concentrations of salt, or TE (pH7.4-8.0) (TE solutions are prepared from a stock of Tris.HCl, pH7.0-9.0, and a stock of EDTA, pH8.0, to yield an aqueous solution consisting of 10mM Tris.HCl and 1mM EDTA).

The methods according to the third and fourth embodiments of the invention are substantially the same as the methods according to the first and second embodiments, respectively, except that the first two methods are suitable for use with microorganisms. For the first step of the method of the third and fourth embodiments, any microorganism suitable for lysis in step (3) of the method can be used for the whole procedure. But typically the microbial culture is a bacterial culture. Microbial cultures are prepared using conventional methods and conditions. As with the previous embodiments, the culture used in the method may be an otherwise produced culture sample or, as described in detail below, may be produced in a device comprising a hollow membrane filter. Thus, in the latter case, the culture is prepared in situ.

The lysis solution of step (3) of the method of the third and fourth embodiments may also comprise a buffer containing a chaotropic agent, with or without a detergent. The above defined chaotropic agents and detergents can also be used for extracting plasmid DNA from microorganisms. Preferred chaotropic agents include guanidine hydrochloride and guanidine thiocyanate at concentrations of 4-6M and 3-5M, respectively. Preferred lysis solutions include:

6M guanidine hydrochloride

0.75％TritonX-100^TM

200mM HEPES (pH6.5) or 100mM Tris.HCl (pH 6.4).

The microorganisms are incubated in the presence of the lysis solution for a sufficient period of time to release large amounts of plasmid DNA, but the amount of chromosomal DNA and other cellular material released is minimized. The incubation time also depends on the type of microorganism. For bacteria such as E.coli, incubation times of 3-15 minutes are suitable.

As in the previous embodiments, steps (3) and (4) of the methods of the third and fourth embodiments may be repeated to improve the yield of plasmid DNA. It will further be appreciated that other detailed methods of the first and second embodiments may also be used with the methods of the third and fourth embodiments.

Now describing a fifth embodiment, the container of the device is generally an elongated cylinder. The container may be any volume, but a volume of about 2 ml is preferred for a device for automated procedures. Typically the hollow membrane filter is no more than about 25% of the volume of the cartridge and may comprise a number of fingers or webs extending into the container or a single cylindrical member extending axially within the container.

The seal for sealing the closed end of the device may be a pull-off foil seal or a cap that seals the end in a screw-fit or friction-fit manner.

In a preferred embodiment, the device also includes a removable seal at the open end of the container. Typically the enclosure is a gas permeable lid to allow aerobic growth of the microorganisms.

It will be appreciated that the device may be configured to allow the addition of culture medium. The desired microorganism is inoculated into the culture medium and the device is incubated under suitable conditions to culture the cells. The culture is then treated according to the extraction procedure of the first embodiment.

In addition, the device can be used to culture virulent phages (e.g., M13 or lambda). The phage is inoculated with the host microorganism. After culturing, the phage can be isolated from the host microorganism by filtration through a hollow membrane filter to extract the culture medium containing the phage particles.

It will also be appreciated that the device of the invention may be used to extract DNA directly from cells, i.e.it is not necessary for the extraction procedure that the cells are cultured in situ.

The device of the fifth embodiment is preferably made from materials that are resistant to organic solvents and conventional sterilization techniques. Suitable materials include polypropylene, polystyrene and acrylate plastics.

The apparatus of the sixth embodiment generally comprises a linear combination of the apparatus of the fifth embodiment wherein each container is an elongated cylinder. Preferably, the device has 8 containers configured to correspond to the wells of a standard 8 x 12 well microtiter plate, which makes the device useful for automated operation.

The plurality of containers in the device combination of the sixth embodiment may be connected together by means of a frangible or solid web. Alternatively, a portion of the wall of one container may abut a portion of the wall of an adjacent container.

It is preferred that the devices of the fifth and sixth embodiments are modified at the open end of the vessel to engage a filtration device containing ion exchange media. This allows the lysis solution containing plasmid DNA to be transferred directly to the ion exchange medium, thereby facilitating purification of the DNA. Once the extracted DNA is bound to the ion exchange medium, the device is disconnected from the filtration device and the remaining DNA purification steps are carried out according to steps (6) and (7) of the second and fourth embodiments.

A particularly preferred way of using the device of the sixth embodiment is in combination with a manifold adapted for connection to a vacuum pump. It is preferred that the manifold comprises a tray having a lid with a plurality of apertures. Typically the lid has at least 8 apertures to receive the linear combination of the devices of the sixth embodiment. Preferably, the lid has 96 wells in a microtiter plate configuration.

When used in conjunction with a manifold, the device is inserted into the aperture of the manifold cap. A plug is applied to the unused cap to allow for the use of negative pressure. Then, if necessary, in steps (1) to (3) of the methods of the first to fourth embodiments, the filtration step is completed by vacuum-pumping through the manifold.

If it is desired to collect the extracted DNA, a microtiter plate is mounted on the manifold plate to collect in the manifold well the DNA-containing lysis solution aspirated from the device assembly.

The manifold may also be connected to the device of the sixth embodiment for purification of the extracted DNA. After performing steps (1) - (3) of the second and fourth embodiments, an ion exchange filtration device can be inserted between the device assembly and the manifold cover to perform step (4). Then the removal device performs steps (5) and (6) in combination, and finally a microtiter plate is mounted on the manifold plate to collect the DNA eluted during step (7).

It will be appreciated that the process described above can automatically control the release of reagents used in carrying out the various steps of the extraction and purification process, the automatic control applying a negative pressure on the manifold.

The kit of the seventh embodiment may include reagents for performing the respective steps of the extraction and purification processes, and/or a filtering device for the purification step, in addition to the device for extracting DNA.

The kit may also include a combination of devices connected to the vacuum manifold. The kit as described above may further comprise a combination of filtration devices.

Brief Description of Drawings

FIG. 1 is a cross-sectional side view of an apparatus comprising a single culture vessel and a filter. The figure also shows the filter used for DNA purification.

FIG. 2 is a cross-sectional side view of an apparatus comprising a plurality of culture vessels and filters, showing the DNA purification filters attached.

Fig. 3 is a plan view from above of the vacuum manifold.

FIG. 4 is a cross-sectional view of a vacuum manifold.

FIGS. 5-10 depict ethidium bromide-stained gels for analysis of DNA samples from various extraction steps.

Best mode for carrying out the invention and other modes

The apparatus of the present invention will first be described with reference to the accompanying drawings. With respect to fig. 1, there is shown a cylindrical member 2 comprising means for receiving a culture medium 3. The device contains a closed end 4 comprising a hollow membrane filter 5. A seal in the form of a cap 6 may be mounted to the closed end 4 in a friction fit to prevent liquid within the device being lost through the hollow membrane filter.

The device 1 has an open end 7 through which the substance is added to the container. A cap 8 with an air-permeable disc is capped at the open end if necessary to maintain sterility. The ventilation of the cover enables the microorganism to ferment aerobically.

The overall diameter of the device depicted in FIG. 1 is 40mm 8 mm OD and is made of polypropylene or polystyrene.

A funnel-type filtering device 9 with a silica mesh 10 therein is shown below the device 1. From this figure it can be seen that the diameter of the tip 4 is reduced so that the tip 4 enters the open end 11 of the device 9. The friction fit between the wall of the cylindrical member 2 and the open end 11 of the device 9 provides a leak-proof seal.

Fig. 2 is now described, in which a linear combination 21 consisting of the 8 devices of fig. 1 is shown. The device is held in assembly by webs, one of which is indicated at 22. For plasmid DNA purification, combination 22 is used in combination with the filtration device combination 23 of FIG. 1.

Fig. 3 depicts the vacuum manifold 31 as viewed from above. The manifold can be seen to have 8 rows of holes, 12 holes in each row.

In fig. 4 a cross-sectional side view of the manifold shown in fig. 3 is provided. The cover 32 and the disc 33 can be seen. Means 34 are provided for connection with a vacuum pump indicated by arrow 35. The manifold 31 of fig. 3 and 4 is drawn on a different scale than that of fig. 2 of the apparatus.

The configuration of the manifold depicted in FIGS. 3 and 4 facilitates extraction of plasmid DNA. In this configuration, the filtration device assembly 23 of fig. 2 is interposed between the assembly 21 and the manifold 31. The funnel-shaped portion of each strainer has a neck 24 that fits into the bore of the manifold cap 32 and forms a seal.

It will be appreciated that the combination 23, when secured thereto, may occupy all of the apertures of a row of the cover 32. Device assembly 21 can then be affixed to assembly 23 for plasmid DNA purification. Through assemblies 21 and 23, which are secured to manifold cap 32, liquid is drawn from each container of the assembly of devices through the silica filter and into tray 33.

It will also be appreciated that a microtiter plate may be placed in chamber 36 of manifold 31 in order to collect liquid passing through the well into a particular well of the microtiter plate.

The following are non-limiting examples. Example 1

Extraction and purification of plasmid DNA

In this series of experiments, the effect of various lysis solutions was evaluated using the method of the second embodiment.

Coli DH10B carrying plasmid pGem5 Zf-was inoculated into LB (Luria Bertani) medium and cultured overnight with shaking at 37 ℃ according to standard procedures. 1 ml of overnight incubated sample was added to 0.2 micron Dynagard with the aid of a 5 ml syringe as funnel^TMME hollow membrane filtration device. The glass fiber filter device was secured to the outlet of the hollow membrane filter device to form a filter assembly. Such filtration devices are known to those skilled in the art and are commercially available.

Cells can be fixed to the hollow membrane filter by pulling a vacuum at the outlet of the filter assembly. To lyse the immobilized cells and release the plasmid DNA, 1 ml of lysis solution was added to the filtration pool and allowed to incubate with the immobilized cells for 5 minutes at room temperature. The lysis solution tested contained a chaotropic agent (guanidine hydrochloride, guanidine thiocyanate or sodium iodide) and a detergent (TritonX-100)^TMOr Brij58^TMSodium deoxycholate). At this step, plasmid DNA is released from the cells and into a solution that binds the DNA to the glass fiber filter. The solution was then pooled through a filter, i.e., through a hollow fiber membrane filter and then through a glass fiber filter-thereby immobilizing the plasmid DNA to the glass fiber filter.

The hollow fiber filter portion of the filter collection was removed and the glass fiber filter was retained prior to elution of plasmid DNA in low salt buffer. The filter was then washed three times under vacuum with 1 ml of 80% isopropanol, 10mM HEPES (ph 6.5). 100 microliters of a low saline solution or TE was added to a vacuum-dried glass fiber filter under vacuum to elute the purified DNA.

Plasmid DNA purified by the above procedure was subjected to agarose gel electrophoresis and DNA bands were detected by ethidium bromide staining. For comparison, pGem5 Zf-prepared by alkaline lysis of E.coli was also analyzed. The alkaline lysis method of the plasmid DNA preparation used is described in Sambrook et al (see above).

The stained gel is depicted in FIG. 5, lanes 2-9 loaded with 25% of the total plasmid amount produced by 1 ml of bacterial culture. The following samples were analyzed: lane 1, EcoRI digested Spp-1 molecular weight marker; lane 2, control of purified plasmid pGem5 Zf-obtained using alkaline lysis; lane 3, using 3.2M guanidine thiocyanate and 0.5% Triton X-100^TM(iii) cleaved prepared pGem5 Zf-; lane 4, using 3.2M guanidine thiocyanate and 0.5% Brij58^TMAnd 0.2% sodium deoxycholate cleaved prepared pGem5 Zf-; lane 5, using 4.8M guanidine hydrochloride and 0.5% Triton X-100^TM(iii) cleaved prepared pGem5 Zf-; lane 6, using 4.8M guanidine hydrochloride and 0.5% Brij58^TMAnd 0.2% sodium deoxycholate cleaved prepared pGem5 Zf-; lane 7, using 4.5M sodium iodide, 90mM sodium sulfite and 0.5% Triton X-100^TM(iii) cleaved prepared pGem5 Zf-; lane 8, using 4.5M sodium iodide, 90mM sodium sulfite, 0.5% Brij58^TMAnd 0.2% sodium deoxycholate cleaved pGem5 Zf-.

As is clear from FIG. 5, the lysis solution tested in the method of the second embodiment can recover plasmid DNA with high purity, although it does not provide as high a yield as the alkaline lysis procedure. Contains 4.8M guanidine hydrochloride and 0.5% TritonX-100^TMThe lysis solution of (2) appears to be particularly effective. Example 2 detection of lysis conditions

Utilizes TritonX-100 containing 4.8M guanidine hydrochloride and various concentrations^TMThe lysis solution of (2) was subjected to further experiments. Various lysis conditions were also tested. Other experimental conditions and procedures are described in example 1. A25% sample of the total plasmid yield from 1 ml of bacterial culture was analyzed by agarose gel electrophoresis.

FIG. 6 shows the results of agarose gel analysis. The following samples were analyzed: lane 1, Spp-1 molecular weight marker digested with EcoRI; lanes 2 and 3, control using purified plasmid pGem5 Zf-obtained by alkaline lysis; lane 4, using a composition containing 1% TritonX-100^TMThe lysis solution lyses the prepared pGem5 Zf-for 5 minutes; lane 5, using a composition containing 2% Triton X-100^TMLysis solutionpGem5 Zf-prepared by lysis in solution for 5 minutes; lane 6, using a composition containing 1% TritonX-100^TMThe lysis solution was incubated for two 5 min to lyse the prepared pGem5 Zf-; lane 7, using a composition containing 2% Triton X-100^TMThe lysis solution was incubated for two 5 min to lyse the prepared pGem5 Zf-; lane 8, 1% Triton X-100 at constant flow rate under vacuum^TMThe lysis solution lyses the prepared pGem5 Zf-for 5 minutes; and lane 9, using a flow rate containing 2% TritonX-100 under vacuum at a constant flow rate^TMThe lysis solution lyses the prepared pGem5Zf for 5 minutes.

As can be seen from the results shown in FIG. 6, the lysis solutions and lysis conditions used in lanes 6 and 7 provided plasmid DNA with a yield and purity essentially equivalent to the alkaline lysis procedure. Example 3

Effect of washing on plasmid yield

A series of experiments were performed to evaluate the effect of washing the bacterial cells prior to the lysis step. The wash solution contained 16.5% sucrose, 36mM Tris.HCl (pH8.0) and 55mM EDTA, and the bacterial cells were incubated in the wash solution at room temperature for various periods of time. By adding 4.8M guanidine hydrochloride, 1% TritonX-100^TMAnd 160mM HEPES (pH6.5), the cells were incubated at room temperature for 5 minutes to effect lysis of the bacterial cells. Other experimental conditions and procedures are described above in example 1. As in the previous examples, a 25% sample of the total plasmid yield from 1 ml of bacterial culture was analyzed by agarose gel electrophoresis.

FIG. 7 depicts the results of agarose gel electrophoresis, in which the following samples were analyzed: lane 1, EcoRI digested Spp-1 molecular weight marker; lane 2, control of purified plasmid pGem5 Zf-obtained using alkaline lysis; lane 3, pGem5 Zf-incubated with wash solution for 5 min, then lysed; lane 4, pGem5 Zf-incubated with wash solution for 30 min, then lysed; lane 5, pGem5 Zf-incubated with wash solution for 5 min, then lysed; lane 6, incubation with wash solution for 30 min, then cleaved pGem5 Zf-.

During the preparation of plasmid DNA analyzed in lanes 3 and 4, the solution was aspirated by the filtration device at-2 kPa, whereas during the preparation of plasmid DNA analyzed in lanes 5 and 6, -13kPa was used.

FIG. 7 shows that the method described in the above embodiment allows the yield and purity of plasmid DNA preparations to be at least equivalent to conventional procedures such as alkaline lysis procedures. The great advantage of the described implementation is that the method can be implemented using a single set of equipment and does not require the transfer of solutions containing DNA. Example 4 extraction and purification of genomic DNA from blood

In a series of experiments, the effect of various lysis solutions was evaluated using genomic DNA recovered from bovine blood.

To 400 microliters of TE was added 100 microliters by volume of bovine blood containing 0.1% (w/v) aqueous EDTA as an anticoagulant. This provides a means to lyse red blood cells present in the blood (heme lysis). The solution was added to 5cm²0.2 micron polypropylene hollow membrane filter pre-wetted with 100% ethanol so that the solution flows through the filter. Filters of this type are commercially available from other materials such as mixtures of cellulose acetate and nitrocellulose, and function in the same manner. The glass fiber filter device was secured to the outlet of the hollow membrane filter device to form a filter assembly.

The cells containing nuclei can be fixed to the hollow membrane filter from the heme-lysed blood by pulling a vacuum at the outlet of the filter assembly. To lyse the immobilized cells and release the genomic DNA, 1 ml of lysis solution was added to the filter pool and allowed to incubate with the immobilized cells for 5 minutes at room temperature. The lysis solution tested contained 4M guanidine thiocyanate, 5% TritonX-100^TM0.1M sodium acetate (pH6.5) and urea at a concentration of from 0.5M to 4M. At this step, genomic DNA is released from the nucleus of the cell and into a solution that binds the DNA to the glass fiber filter. This step was repeated 3 times to completely release the DNA.

The filter was then washed three times under vacuum with 1 ml of 80% isopropanol, 10mM Tris.HCl (pH 6.4). 100 microliters of a low salt solution of TE (pH8.5) was added to the glass fiber filter under vacuum to elute the purified genomic DNA.

The purified genomic DNA was subjected to agarose gel electrophoresis and the DNA bands were detected by ethidium bromide staining. For comparison, the use of Progen Progenome was also analyzed^TMI genomic DNA purification kit (available from Progen industries, Ltd., 2806 IpswichRoad, Darra, Queensland 4076, Australia) was used for analysis.

Fig. 8 depicts a stained gel. Lanes 2-7 are loaded with 25% of total genomic DNA produced from 100 microliters of bovine blood. The following samples were analyzed: lane 1, EcoRI digested Spp-1 molecular weight marker; lane 2, using Progeneme^TMI control of purified genomic DNA; lane 3, genomic DNA prepared by lysis with lysis solution containing 0.5M urea; lane 4, genomic DNA prepared by lysis with lysis solution containing 1M urea; lane 5, genomic DNA prepared by lysis with lysis solution containing 2M urea; lane 6, genomic DNA prepared by lysis with lysis solution containing 3M urea; lane 7, genomic DNA prepared by lysis with lysis solution containing 4M urea.

In the absence of a supply such as Progeneome^TMI genomic DNA purification kit as high yield, the lysis solution tested provided a highly pure genomic DNA preparation. Contains 4M guanidine hydrochloride and 5% TritonX-100^TMLysis solutions of 0.1M sodium acetate pH5.0 and urea at a concentration of 3M appear to be particularly effective. Example 5 comparison of culture methods

Coli DH10B carrying plasmid pMW5 was inoculated into LB medium and incubated overnight at 37 ℃ with shaking according to standard procedures. (pMW5 was constructed by Progen industries, Inc. and includes a partial lambda phage in pUC 19). To examine the various culture methods, the strain was also inoculated into LB medium and incubated with or without aeration at 0.2 μm (5 cm)²) The culture was carried out overnight at 37 ℃ in a polypropylene hollow fiber filter unit. Aeration of the culture can be achieved by applying adjustable compressed air to the outlet of the hollow fiber filtration device. Tong (Chinese character of 'tong')Gas is a visible form of gas bubbles that pass through the medium.

After overnight incubation, 250 microliters of standard culture was applied to a hollow membrane filtration device that was previously wetted with 100% ethanol to allow solution flow through the filter. The hollow membrane filter is the same type of device that employs in situ culture as described above. Therefore, subsequent analyses were performed with standard samples, the overnight culture in situ was removed from the hollow membrane filtration device and the 250 microliter portion was added to a similar fresh device similar to that pre-wetted with 100% ethanol. In practice, however, the in situ culture is filtered in a hollow membrane filtration unit for cultivation and is not transferred to a fresh unit. The glass fiber filter device was secured to the outlet of the hollow membrane filter device to form a filter assembly.

Cells can be fixed to the hollow membrane filter by pulling a vacuum at the outlet of the filter assembly. Prior to bacterial lysis and release of plasmid DNA, 1 ml of a solution containing 16.5% sucrose, 40mM Tris.HCl (pH8.0) and 60mM EDTA was used to wash the cells by applying a solution containing 6M guanidine hydrochloride, 0.1M Tris.HCl (pH6.4), 0.75% Triton X-100^TMThe solution of (2) was incubated with the fixed cells at room temperature for 5 minutes for lysis of the bacterial cells and release of plasmid DNA. Then the solution containing plasmid DNA was aspirated from the filter assembly under vacuum, thereby immobilizing the plasmid DNA to the glass fiber filter. The lysis step is then repeated to maximize recovery of plasmid DNA.

Before elution of plasmid DNA from the glass fiber filter, the hollow fiber filter portion of the filtration assembly was removed, leaving the glass fiber filter. The latter filter is then washed three times under vacuum with 1 ml of 80% isopropanol, 10mM Tris.HCl (pH 6.4). A volume of 100. mu.l of a low salt solution of TE (pH8.5) was added to a vacuum-dried glass fiber filter under vacuum to elute the purified DNA.

Plasmid DNA purified by the above procedure was subjected to agarose gel electrophoresis and DNA bands were detected by staining with ethidium bromide.

The stained gel is depicted in fig. 9. Lanes 2-4 are loaded with 25% of the total plasmid produced from 250 microliters of bacterial culture. The following samples were analyzed: lane 1, Spp-1 molecular weight marker digested with EcoRI; lane 2, plasmid DNA purified from bacteria cultured overnight at 37 ℃ in a 0.2 micron hollow fiber filter unit without aeration; lane 3, plasmid DNA purified from bacteria cultured overnight at 37 ℃ in a 0.2 micron hollow fiber filter unit with aeration; lane 4, plasmid DNA purified from bacteria cultured overnight at 37 ℃ according to standard methods.

As is clear from FIG. 9, the bacteria cultured in the hollow fiber filtration device under aeration did not hinder the recovery of the plasmids. This result confirmed that it was possible to culture the bacteria in the same hollow membrane filter used for the subsequent DNA extraction step. Example 6 extraction of DNA from cultured animal cells

A series of experiments were performed to evaluate the genomic DNA recovered from cultured mammalian cells using the extraction procedure of the present invention.

A suspension of human intestinal cancer cells (HCT-116; ATCC accession number CCL 247) cultured in McCoys 5a medium was added to 0.2 μm Dynagard by means of a 5 ml syringe as a funnel^TMME hollow fiber filtration device. The glass fiber filter device was secured to the outlet of the hollow membrane filter device to form a filter assembly.

Cells were fixed from solution to the hollow fiber filter by applying a vacuum at the outlet of the filter assembly. To lyse the cells and release the genomic DNA, 1 ml of lysis solution was added to the filter pool and allowed to incubate with the immobilized cells for 5 minutes at room temperature.

Lysis solution contained 4M guanidine thiocyanate, 5% TritonX-100^TM0.1M sodium acetate (pH5.0) and 3M urea. At this step, genomic DNA is released from the cells and into a solution that binds the DNA to the glass fiber filter. This step was repeated 3 times to maximize the release of DNA from the fixed cells.

Before elution of genomic DNA from the glass fiber filter, the hollow fiber filter portion was removed, leaving a collection of glass fiber filters. The filter was then washed three times under vacuum with 1 ml of 80% isopropanol, 10mM Tris.HCl (pH 6.4). A volume of 100. mu.l of a low salt solution of TE (pH8.5) was added to the glass fiber filter under vacuum to elute the purified genomic DNA.

Genomic DNA was subjected to agarose gel electrophoresis and DNA bands were detected by staining with ethidium bromide.

The stained gel is depicted in fig. 10. Lanes 2-5 are loaded with 25% of total genomic DNA yield. The following samples were analyzed: lane 1, Spp-1 molecular weight marker digested with EcoRI; lane 2, genomic DNA prepared from lysis of human intestinal cancer cells from 1 ml of suspension; lane 3, genomic DNA prepared from human intestinal cancer cells lysed from 2 ml suspension; lanes 4 and 5, genomic DNA prepared by lysing human intestinal cancer cells from 5 ml of suspension.

The results depicted in fig. 10 show that the extraction method of the present invention can be effectively applied to cultured animal cells.

It will be appreciated that many modifications may be made to the method and apparatus described above without departing from the broad ambit and scope of the invention as defined in the appended claims.

Claims

1. A method for extracting DNA from a cell suspension, said method comprising the steps of:

1) the cell suspension is added to a filtration device having a hollow membrane filter,

2) if necessary, filtering to remove the medium suspending the cells;

4) the lysis solution containing the DNA was filtered.

2. The method of claim 1, wherein the DNA is genomic DNA.

3. The method of claim 1, wherein the cell suspension is cultured animal cells.

4. The method of claim 1, wherein the cell suspension is a body fluid selected from the group consisting of blood, lymph, semen and urine.

5. The method according to claim 1, further comprising a step of washing the cells collected on the hollow fiber filter in step (2).

6. The method of claim 1, wherein the lysis solution comprises a buffered solution of a chaotropic agent.

7. The method of claim 6, wherein the chaotropic agent is selected from the group consisting of guanidine hydrochloride, guanidine thiocyanate, sodium iodide, and sodium perchlorate.

8. The method of claim 6, wherein the lysis solution further comprises a detergent.

9. The method of claim 8, wherein the detergent is selected from Tween ™^TM20，TritonX-100^TM，Nonidet^TM P-40，Brij58^TMSodium deoxycholate, N-lauroylsarcosine.

10. The method of claim 9, wherein the lysis solution comprises 4M guanidine thiocyanate, 0.1M sodium acetate (ph5.0), 5% triton x-100^TMAnd 3M urea.

11. The method of claim 1, wherein the step of filtering the lysis solution is accomplished by applying a vacuum at an outlet of the device.

12. The method of claim 1, comprising repeating steps (3) and (4).

13. The method of claim 1, wherein the cells are cultured on the cell filtration device.

14. The method of claim 13, comprising supplying air into the device.

15. The method of claim 14, wherein the air is supplied to the culture through the hollow membrane filter.

16. A method for extracting DNA from a cell suspension, the method comprising the steps of:

2) if necessary, filtering to remove the medium suspending the cells;

4) filtering the lysis solution containing the DNA;

5) adding the filtrate of step (4) to an ion exchange medium;

7) washing the ion exchange medium with a second solution to elute the DNA.

17. The method of claim 16, wherein the ion exchange media is an inert substrate substituted with diethylaminoethyl, quaternary aminoethyl, or quaternary ammonium groups.

18. The method of claim 16, wherein the ion exchange media is silica.

19. The method of claim 16, wherein the first solution is an alcohol solution that does not dissolve the DNA.

20. The method of claim 16, wherein the second solution is an aqueous solution that dissolves the DNA.

21. The method of claim 16, wherein the filtration device is directly bonded to a device comprising the ion exchange media.

22. A method for extracting plasmid DNA from a microbial culture, the method comprising the steps of:

1) adding a plasmid-carrying microbial culture to a filtration device having a hollow membrane filter;

2) if necessary, the medium is removed by filtration;

4) the lysis solution containing the plasmid DNA was filtered.

23. The method of claim 22, wherein the microbial culture is a bacterial culture.

24. The method of claim 22, wherein the microorganism is cultured in the device.

25. The method of claim 22, further comprising the step of washing the cells collected on the hollow fiber filter in step (2).

26. The method of claim 22, wherein the lysis solution comprises a buffered solution of a chaotropic agent.

27. The method of claim 26, wherein said chaotropic agent is selected from the group consisting of guanidine hydrochloride, guanidine thiocyanate, sodium iodide, and sodium perchlorate.

28. The method of claim 26, wherein the lysis solution further comprises a detergent.

29. The method of claim 28, wherein the detergent is selected from Tween^TM20，TritonX-100^TM，Nonidet^TM P-40，Brij58^TMSodium deoxycholate, N-lauroylsarcosine.

30. The method of claim 29, wherein the lysis solution comprises 6M guanidine hydrochloride, 0.75% Triton X-100^TMAnd 200mM HEPES (pH6.5) or 100mM Tris. HCl (pH 6.4).

31. The method of claim 22, wherein the step of filtering the lysis solution is accomplished by applying a vacuum at an outlet of the device.

32. The method of claim 22, comprising repeating steps (3) and (4).

33. The method of claim 22, wherein the cells are cultured on the cell filtration device.

34. The method of claim 33, comprising supplying air into the device.

35. The method of claim 34, wherein the air is supplied to the culture through the hollow membrane filter.

36. A method for extracting plasmid DNA from a microbial culture, the method comprising the steps of:

2) if necessary, the medium is removed by filtration;

4) filtering the lysis solution containing the plasmid DNA;

5) adding the filtrate of step (4) to an ion exchange medium;

37. The method of claim 36, wherein the ion exchange medium is an inert substrate substituted with diethylaminoethyl, quaternary aminoethyl, or quaternary ammonium groups.

38. The method of claim 36, wherein the ion exchange media is silica.

39. The method of claim 36, wherein the first solution is an alcohol solution that does not dissolve the DNA.

40. The method of claim 36, wherein the second solution is an aqueous solution that dissolves the DNA.

41. The method of claim 36, wherein the filtration device is directly bonded to a device comprising the ion exchange media.

42. An apparatus for culturing cells and extracting DNA therefrom, the apparatus comprising:

a movable seal sealing said closed end.

43. The device of claim 42, wherein the container is a cylinder having a volume of about 2 milliliters.

44. The apparatus of claim 43, wherein the hollow membrane filter is a single cylindrical piece extending axially within the vessel.

45. A device as claimed in claim 42 wherein the seal for sealing the closed end of the device is a removable foil seal or a cap which seals the end in a screw or friction fit.

46. The device of claim 42, further comprising a removable seal at the open end of the container.

47. The device of claim 46, wherein the movable seal is a gas-permeable cover.

48. The device of claim 42, wherein the closed end of the vessel is adapted to secure a filtration device comprising ion exchange media.

49. The device of claim 42, wherein the device container is made from a plastic selected from the group consisting of polypropylene, polystyrene, and acrylate.

50. A device for culturing cells and extracting DNA therefrom, the device comprising a plurality of devices according to claim 42.

51. A device for culturing cells and extracting DNA therefrom, the device comprising a linear combination of a plurality of devices of claim 42, wherein each container is an elongated cylinder.

52. The device of claim 51, which is comprised of 8 containers to correspond to the wells of a standard 8 x 12 well microtiter plate.

53. The device of claim 52, wherein the containers are connected by means of a frangible mesh.

54. The apparatus of claim 52, wherein at least a portion of a wall of one container abuts at least a portion of a wall of an adjacent container.

55. The device of claim 52, wherein the closed end of each receptacle in the device combination is adapted to secure a filtration device comprising ion exchange media.

56. A kit for culturing cells and extracting DNA therefrom, the kit comprising at least one device selected from any one of claims 42, 50 or 51.

57. The kit of claim 56, further comprising reagents for extracting said DNA.

58. The kit of claim 56, further comprising a vacuum manifold adapted to engage the device of claim 50 or 51.

59. The kit of claim 56, further comprising a means for ion exchange purification of DNA.

60. The kit of claim 59, wherein the ion exchange device comprises a combination of devices adapted to engage claim 50 or 51.

61. The kit of claim 56, further comprising a vacuum manifold adapted to engage the device of claim 50 or 51 directly or sequentially connected to the ion exchange device of claim 60.

62. The kit of claim 61, wherein the vacuum manifold is adapted to engage a standard 8 x 12 well microtiter plate.