WO2009014415A1

WO2009014415A1 - A method, kit and apparatus for extracting biological materials

Info

Publication number: WO2009014415A1
Application number: PCT/MY2008/000059
Authority: WO
Inventors: Beow Chin Yiap
Original assignee: Beow Chin Yiap
Priority date: 2007-07-23
Filing date: 2008-06-26
Publication date: 2009-01-29
Also published as: MY157651A

Abstract

The present invention relates to a method of extracting biological materials from biological samples. A series of solutions used in the method of the present invention are customizable into different formulations to optimize the extraction conditions and are usable in the extraction of different biological materials such as genomic DNA, plasmid DNA, RNA or protein from a wide range of organisms.

Description

A method, kit and apparatus for extracting biological materials

Field of Invention

The present invention relates to a method of extracting biological materials such as nucleic acids or proteins from biological samples, kit and apparatus thereof.

Background of the Invention

Extraction, isolation and purification of nucleic acids or proteins are important steps in many biochemical and diagnostic procedures. A number of methods are known to have been used for the extraction, isolation and purification of nucleic acids or proteins from test samples. Generally, such methods have to go through a sequence of procedures and solutions are used in the performance of such procedures. The solutions used in the existing methods are normally premixed and are prepared at certain concentrations only. Such premixed solutions are normally pre-set to extract only a specific nucleic acid such as DNA, RNA, or plasmid DNA from a specific sample or from various samples of the same species. For example, a set of premixed solutions in certain concentrations is used to extract either DNA, RNA, plasmid DNA or protein from a particular organism only.

There are a lot of commercial extraction kits available in the market for the extraction of nucleic acids or proteins from test samples. These extraction kits are normally committed to a narrow range of specific and related organisms without the flexibility to adjust or alter the quantity of the sample used. These extraction kits are provided with a set of solutions which are premixed at their required concentrations and are designed to obtain the target nucleic acid from a particular organism only.

As such, a user who wishes to extract different target nucleic acids from a particular organism or wishes to extract the same target nucleic acid from two different organisms may need to purchase two or more extraction kits with different sets of extraction solutions respectively to perform the extractions.

Furthermore, most of the commercial kits available in the market involve the column-based technology. These column-based kits are normally expensive and time consuming to perform. In addition, the plastic columns utilized in these kits are both an environmental hazard and wastage of natural resources as they are non-reusable and non-recyclable. Such kits also involve the usage of dangerous chemicals such as phenol, chloroform, caesium chloride, and ammonium sulfate that might be harmful to the user.

Summary of Invention

There is a need to provide improved methods for the extraction, isolation and purification of nucleic acids or proteins. It is therefore the present invention provides a method for the same whereby the same series of solutions are customizable into several formulations to optimize the extraction conditions. The customized formulations are usable for a wide range of organisms to obtain their biological materials.

A particular area of improvement of the present invention is the usability of the same series of chemical solutions for the extraction of biological materials from a wide range of organisms. Furthermore, there is a flexibility to adjust or alter the quantity of biological sample used in the extraction to suit the specific needs of every extraction process.

The present invention also provides a method of extracting biological materials from biological samples whereby the chemical solutions used are prepared from a group of pre-determined chemicals in their solid or liquid forms and said chemicals are able to be customized according to the requirements of each extraction. The present invention provides a method of extracting biological materials from biological samples whereby the group of p re-determined chemical solutions can be customized according to the target biological materials to be extracted and the biological sample from which the target biological materials are extracted.

The present invention also includes a series of solutions or a group of chemicals for the preparation of that series of solutions. The said series of solutions or group of chemicals is packaged as a kit for the extraction of a wide variety of biological materials from various biological samples.

In one embodiment, the present invention provides a method of extracting biological material from a biological sample via a sequence of procedures which involves the mixing of said biological sample with a series of solutions at their effective concentrations wherein said series of solutions is obtainable from a group of pre-determined chemicals which are able to be customized according to the requirements of the target biological material to be extracted and the biological sample used.

The abovementioned solutions are obtainable from a group of pre-determined chemicals comprises of alkali metal chloride salt, detergent, alkali metal hydroxide, concentrated weak organic carboxylic acid, polyethers, alkali metal acetate salt, chelating agent, and RNase inhibitor.

According to the preferred embodiment of the present invention, the alkali metal chloride salt is sodium chloride; the detergent is sodium dodecyl sulphate (SDS) ; the alkali metal hydroxide is sodium hydroxide; the concentrated weak organic carboxylic acid is acetic acid preferably glacial acetic acid; the polyethers is polyethylene glycol (PEG); the alkali metal acetate salt is any one or combination of sodium acetate and potassium acetate; the chelating agent is ethylenediaminetetra acetic acid (EDTA) and the RNase inhibitor is diethylpyrocarbonate (DEPC). The method of extracting biological material from a biological sample according to the present invention comprises the following steps: a), adding cellular disruption solution to said biological sample; b). incubating the disrupted sample from step a at a p re-determined temperature; c). adding separation solution to the incubated sample from step b to form a mixture; d). spinning the resulting mixture from step c in a pre-determined condition to obtain supernatant; e). adding extraction solution to the supernatant obtained from step d to form a mixture; f). spinning the resulting mixture from step e in a pre-determined condition to obtain the pellet which comprises the target biological material; wherein said cell disruption solution, separation solution and extraction solution are obtainable from the aforementioned series of chemicals and are flexible to be customized into different formulations according to the requirements of the extraction to obtain the highest yield and purity.

In another embodiment, the present invention provides a kit for the extraction of biological materials from biological samples and the kit comprises at least cellular disruption solution; separation solution and extraction solution. The cell disruption solution, separation solution and extraction solution of the present invention are obtainable from the aforementioned group of chemicals. By customizing the same series of chemicals at appropriate concentrations and volumes, various biological materials such as DNA, RNA, plasmid DNA or proteins can be extracted from a wide range of organisms.

In a further embodiment, the present invention provides an apparatus for dispensing solutions required for the extraction of biological material at the preprogrammed or user-defined concentrations and volumes.

The present invention also provides an automated apparatus to carry out the aforementioned method of the present invention. Brief description of drawings

For a better understanding, the present invention will be described with reference to the accompanying drawings in which:

Figure 1 illustrates an apparatus for the dispensing of solutions required in the extraction of biological materials according to one embodiment of the present invention;

Figure 2 illustrates an automated apparatus to carry out the method of the present invention;

Figure 3 illustrates gel electrophoresis images of genomic DNA extracted through the method of the present invention from (a) a flower petal; (b) a Gram-negative bacterium; (c) a Gram-positive bacterium; (d) an invertebrate; (e) a vertebrate; (f) an insect; and (g) a mollusc;

Figure 4 illustrates gel electrophoresis images of plasmid DNA extracted through the method of the present invention from (a) a Gram-negative bacterium; and (b) a Gram-positive bacterium;

Figure 5 illustrates a gel electrophoresis image of PCR products obtained from the method of the present invention;

Figure 6 illustrates a gel electrophoresis image of restriction enzyme digestion of plasmid DNA from a Gram-negative bacterium obtained through the method of the present invention

Detailed description of the preferred embodiments

The description in detail for the preferred embodiments of the present invention will now be given with some of them illustrated by the accompanied drawings. While the invention will be described in conjunction with the preferred embodiments, it should be understood that they are not intended to limit the inventior/to those embodiments.

The present invention relates to a method of extracting biological materials from a wide/variety of samples. The chemical solutions used for this purpose can be prepared from a group of pre-determined chemicals in their solid or liquid forms and the chemicals of the present invention are customizable to form different fprmulations required for the extractions of biological materials from a wide variety bf samples. Specifically, the chemical content of the solutions are customizable /according to the requirements of target biological materials and the organism from which the target biological materials are extracted.

The term "biological material" as used herein refers to any target nucleic acid such as genomic DNA, plasmid DNA, RNA or protein of interest.

The term "sample" or "biological sample" as used herein refers to any composition or mixture in which a target nucleic acid or protein of interest may be present, including but not limited to microorganisms, plant or animal materials, food materials, waste materials, materials for forensic analysis, environmental samples, and the like. A biological sample includes any tissue, cell, or extract derived from a living or dead organism which may contain a target nucleic acid or target protein.

The term "extracting" or "extraction" as used herein includes the processes of separating, isolating or purifying of biological materials from a sample or biological sample as defined above.

In one embodiment, the present invention provides a method for extracting biological material from a biological sample via a sequence of procedures which will be further described hereinafter. When carrying out the procedures, a series of solutions are used and the series of solutions are obtainable from a group of pre-determined chemicals. The series of chemicals according to the present invention are customizable into different formulations as required to perform the extractions on a wide range of organisms. The chemicals are customizable into different formulations with different concentrations as desired depending on the target biological materials and the biological samples from which the target biological materials are extracted.

According to the present invention, the group of pre-determined chemicals basically comprises eight chemicals. These eight chemicals include (i) alkali metal chloride salt preferably sodium chloride; (ii) detergent preferably sodium dodecyl sulphate (SDS); (iii) alkali metal hydroxide preferably sodium hydroxide; (iv) concentrated weak organic carboxylic acid preferably acetic acid and more preferably glacial acetic acid; (v) polyethers preferably polyethylene glycol (PEG); (vi) alkali metal acetate salt preferably any one or combination of sodium acetate and potassium acetate; (vii) chelating agent preferably ethylenediaminetetra acetic acid (EDTA) and (viii) RNase inhibitor preferably diethylpyrocarbonate (DEPC).

These chemicals can be mixed according to the requirements of extractions into a series of solutions which comprises basically cellular disruption solution, separation solution and extraction solution. Preferably, the cellular disruption solution comprises about 1 - 5 % weight by volume (w/v) of SDS and about 0.1 - 1 M of EDTA or about 0.5 - 2M of NaCI plus about 0.1 - 1 M of EDTA for the extraction of native protein. The separation solution comprises about 1 - 5M of sodium acetate of which the pH has been adjusted to about 4 - 6 preferably 4.8 by using an appropriate volume of glacial acetic acid. The extraction solution comprises about 20 - 30% weight by volume (w/v) of PEG and 1 - 2M of NaCI.

According to the present invention, the use of the cellular disruption solution is to disrupt the cellular surface of the sample so that the cellular contents which consist the desired biological materials are released. As mentioned above, the cellular disruption solution of the present invention comprises the mixture of SDS and EDTA or NaCI and EDTA for the extraction of native protein. The SDS content therein is a detergent that can denature the proteins on the cell surface which would then lyse the cell to release nucleic acids and proteins. Whereas, EDTA is a chelating agent which will "punch holes" on the cell surface by disrupting the conformation of the surface proteins, leading to cell rupture through osmotic shock to release nucleic acids and proteins. The NaCI which is hypertonic will cause cell rupture through osmotic shock to release nucleic acids and proteins.

Separation solution is used to separate out the contaminant when high speed centrifugation is applied to the mixture of sample and separation solution. The separation solution comprises sodium acetate of which the pH has been adjusted to about 4.8 by adding an appropriate volume of glacial acetic acid. The mixture of sodium acetate and glacial acetic acid will form a low acidic pH salt solution important for the precipitation of denatured biological materials such as protein and genomic DNA if necessary as in plasmid DNA & RNA extraction only The addition of separation solution will cause the contaminant to precipitate leading to the changes of the sample appearance to cloudy. Upon centrifugation and removal of contaminant sediments, the sample will appears to be clear.

Extraction solution is utilized to extract the desired nucleic acids from the aqueous solution that contains residual contaminants. The extraction solution substantially comprises PEG and NaCI. PEG and NaCI at a high concentration will enable precipitation of nucleic acids.

If a user wishes to extract plasmid DNA, an additional denaturing solution which comprises about 1 - 5M of NaOH is applied to the sample prior to the addition of cellular disruption solution. The NaOH is a strong alkaline that can denature many biomolecules at high pH. It is useful in denaturing genomic DNA and proteins for subsequent separation from plasmid DNA in order to obtain pure plasmid DNA.

RNase inhibitor preferably DEPC may be used to deactivate the activity of RNase in destroying RNA.

General protocol for extraction of DNA or RNA according to the present invention Generally, the method for extracting biological material such as DNA or RNA from a biological sample according to the present invention comprises firstly, placing an adequate amount of sample into a tube and followed by adding the cellular disruption solution into the sample and then mix well; secondly, incubating the resulting mixture at about 37⁰C for about 5 - 10 minutes; thirdly, adding separation solution into the sample and mix well by gentle inversion; fourthly, spinning the resulting mixture at about 10,000 - 15,00Og at room temperature for about 1 to 6 minutes; fifthly, pouring the supernatant into a new tube and add a substantially equal volume of extraction solution into the supernatant and then mix well by inversion; sixthly, precipitating the target nucleic acid by spinning the resulting mixture at about 10,000 - 15,00Og at room temperature for about 6 to 10 minutes; lastly, vacuum dry the tube for about 2 - 3 minutes and add 100 - 500μl of distilled water or TE buffer into the tube for storage purposes.

The protocol described above applies generally to the extractions of all kinds of biological materials from various biological samples. However, to obtain a better result, certain steps are needed to be carried out when preparing the samples based on the natural conditions and forms of the samples prior to the above general protocol.

For solid samples such as plant, fungus, animal tissues etc, a lyses process is preferably be carried out prior to the above protocol to provide the sample a maximum surface contact with the solutions. Preferably, the lyses process is performed by physical or chemical means, more preferably, by cutting, ultrasonication, homogenization, or grinding of the samples into powder form at a low temperature to inhibit protein activity. This can be achieved by performing the lyses process in the presence of liquid nitrogen.

As for the samples like yeast, Gram-positive bacteria, adhesion cell culture etc, an appropriate enzymatic treatment is recommended before adding the cellular disruption solution. The enzymatic treatment is the common enzymatic treatment that known in the field. After the enzymatic treatment, the samples are centrifuged to obtain the pellet. The cellular disruption solution is then added into the pellet and this followed by the general protocol above.

For Gram-negative bacteria or suspension cell culture that do not require enzymatic treatment, the samples are centrifuged to get the pellet before adding the cellular disruption solution to the pellet.

Protocol for extraction of plasmid DNA according to the present invention

Extraction of plasmid DNA from a sample may need an additional solution. This additional solution is denaturing solution which comprises NaOH. Denaturing solution is added into cellular disruption solution at a ratio of about 1 :5 and this is then followed by the steps of general protocol as described above.

Protocol for extraction of native protein according to the present invention

The sample preparation protocol for extraction of native protein is similar to the protocol for the extraction of DNA or RNA as described above. However, instead of using the cellular disruption solution comprising SDS and EDTA, cellular disruption solution comprises 0.1 - 1 M of EDTA and 1 - 2M of NaCI is used. The sample prepared based on its natural condition and form as described above is centrifuged at about 10000 - 1500Og at room temperature for about 6 - 15 minutes. The supernatant containing the desired protein is then collected.

Protocol for extraction of denatured protein according to the present invention

The sample preparation protocol for extraction of native protein is similar to the protocol for the extraction of DNA or RNA as described above. However, the cellular disruption solution will be comprised of SDS and EDTA. The sample prepared based on its natural condition and form as described above is centrifuged at about 10000 - 1500Og at room temperature for about 6 - 15 minutes. The supernatant containing the desired protein is then collected. Preparation of solutions

The solutions used in the present invention can be prepared in many ways and according to the preferred embodiments of the present invention, the solutions can be prepared by either (A) preparing the stock solutions for all eight chemicals and then mix the stock solutions according to the extraction requirements to form the required solutions when the solutions are needed or (B) by preparing the ready-to-use solutions. The following are illustrated as examples and not intended to limit the present invention:

A. Preparation of stock solutions

1. SDS (sodium dodecyl sulphate)

• Weigh the corresponding amount of chemicals to make a final concentration of about 2 - 10 % SDS, mix and dissolve in water at about 65°C

• Sterilization is achieved through overnight incubation (a minimum of 4 hours) with DEPC.

2. EDTA (ethylenediaminetetraacetic acid) (pH 6.5 - 8.5)

• Weigh the corresponding amount of chemicals to make a final concentration of about 0.2 - 3 M EDTA, mix and dissolve in water, adjust pH to about 8.0 with NaOH

• Sterilization is achieved through overnight incubation (a minimum of 4 hours) with DEPC and autoclaving.

3. NaOH (Sodium hydroxide)

• Weigh the corresponding amount of chemicals to make a final concentration of about 1 - 5 M NaOH, mix and dissolve in water, • Sterilization is achieved through overnight incubation (a minimum of

4 hours) with DEPC and autoclaving.

4. Sodium acetate (pH 4 - 6) • Weigh the corresponding amount of chemicals to make a final concentration of about 1 - 5 M Sodium acetate, mix and dissolve in water, adjust pH to 4.8 with glacial acetic acid

5. Glacial acetic acid (ready to use, commercially available)

6. NaCI (Sodium Chloride) • Weigh the corresponding amount of chemicals to make a final concentration of about 1 - 5 M NaCI, mix and dissolve in water

7. PEG (Polyethylene glycol)

• Weigh the corresponding amount of chemicals to make a final concentration of about 20 - 60 % PEG, mix and dissolve in water

8. DEPC (Diethylpyrocarbonate) (ready to use, commercially available)

B. Preparation of ready-to-use solutions

a. Cellular disruption solution

i). Cellular disruption solution for extraction of nucleic acids and denatured proteins

• Weigh the corresponding amount of chemicals to make a final concentration of about 1 - 5 % of SDS and about 0.1 - 1 M EDTA, mix and dissolve in water or

ii). Cellular disruption solution for extraction of native proteins • Weigh the corresponding amount of chemicals to make a final concentration of about 0.5 - 2M of NaCI and about 0.1 - 1M EDTA, mix and dissolve in water

For B(a)(i) and B(a)(ii) - Sterilization is achieved through overnight incubation (a minimum of 4 hours) with DEPC and autoclaving.

RNase is to be added by user prior to extraction of genomic or plasmid DNA only.

b. Separation solution

• Weigh the corresponding amount of chemicals to make a final concentration of about 1 - 5 M Sodium acetate with its pH adjusted to 4.8 with the appropriate volume of glacial acetic acid, mix and top-up with water • Sterilization is achieved through overnight incubation (a minimum of

4 hours) with DEPC and autoclaving.

c. Extraction solution

• Weigh the corresponding amount of chemicals to make a final concentration of about 20 - 30 % of PEG and about 1 - 2M NaCI, mix and dissolve in water

d. Denaturing solution [functioning in denaturing genomic DNA during extraction of plasmid DNA process]

• Weigh the corresponding amount of chemicals to make a final concentration of about 1 - 5 M NaOH, mix and dissolve in water

As mentioned above, the group of chemicals according to the present invention are able to be mixed in any other way and amount as deem appropriate depending on the target biological material and the biological sample of which the target biological material extracted from.

As an exapnple, Researcher A who wants to extract genomic DNA from 10ml of bacteria culture can prepare the solutions of cellular disruption solution, separatio'n solution and extraction solution at the concentrations as stated in Table 1 ,below.

Tabled Solutions used for extraction of genomic DNA from bacteria culture

By using the combination of the chemicals and their concentrations as stated in Table 1 , the researcher may use only 0.1 ml of culture instead of 10ml of culture. The researcher can easily scale down his extraction volume while get the same result.

In another scenario, if researcher needs to isolate plasmid DNA, all he needs to do is to prepare an additional solution which is denaturing solution that consists of about 1 - 5M of NaOH. Even if he wishes to obtain RNA or protein from the bacteria culture, the same set of chemicals can be customized to achieve that.

Meanwhile, other researchers who are doing researches on various organisms are able to extract the biological materials from various organisms by using the same series of solutions as listed in Table 1. In order to obtain the satisfactory results, all the researchers need to do is to reformulate the solutions to meet their extraction requirements. For example: i) if the biological sample is of high protein content (plant samples, Gram- positive bacteria) high concentration of SDS can be used and vice versa. ii) if low yield of nucleic acids was form, an increased volume of NaCI and PEG in the extraction solution can be applied to overcome this. iii) if residual of contaminants was found in the supposedly purified nucleic acid sample, the contents of the NaCI and PEG in the extraction solution can be reduced.

By doing so, the same set of solutions according to the present invention can be used to extract various biological materials from a wide range of organisms.

According to another aspect of the present invention, there is provided a kit for extracting and isolating nucleic acids or proteins using the method of the present invention. The kit comprises at least the following: a. cellular disruption solution b. separation solution c. extraction solution as described above wherein said cell disruption solution, separation solution and extraction solution are able to be customized according to the requirements of the extraction and therefore are usable for a wide range of organisms to obtain the high yield.

The extraction kit of the present invention may further comprises denaturing solution for the use in the extraction of plasmid DNA and RNase inhibitor for the use in the extraction of genomic or plasmid DNA.

A further aspect of the present invention provides an apparatus for dispensing solutions required for the extraction of biological materials specially the solutions as described above. The concentrations and volumes of the solutions required can be pre-programmed or customized according to the requirements for the extraction of target biological materials. Referring to the Figure 1 , the apparatus (10) for dispensing solutions according to the present invention comprises a housing (12), a computer controller (not shown) for controlling the apparatus, a plurality of solution drums (14) provided in said housing (12), and a dispensing hose (16) connected to the solution drums (14) via a conduit (not shown) to dispense the solution from said solutions drums (14) into a container when such solution is needed. One wall of the housing (12) further comprises a plurality of selection buttons (18) and at least a display screen (20) to show the command lines, processes etc.

The apparatus (10) for dispensing solutions according to the present invention can be pre-programmed or customized to dispense sufficient solutions needed for the extraction of certain biological material from a specific organism. For example, one of the selection buttons has been pre-programmed to dispense the solutions need for the extraction of genomic DNA from bacteria culture. By pressing the button, the solution needed will be dispensed from the apparatus in a sufficient volume and concentration. The user may use the solution as dispensed from the apparatus directly for the steps required to obtain the target genomic DNA.

Referring now to the Figure 2, the present invention also provides an automated apparatus (100) to carry out the method of the present invention wherein said apparatus comprises of: (a) a computer controller (not shown) for controlling said automated apparatus

(100); (b) a liquid dispensing unit (10) for the dispensing of solutions; (c) a housing (102) having:

(i) at least an input bay (104) for the feeding of sample cassette into the housing (102);

(ii) at least an output bay (106) for the ejection of cassette containing the extracted biological materials from the apparatus; and

(iii) at least a waste container (108) for collection of waste fluid from the extraction process; (d) a mechanical moving part (110) for holding of the sample cassette and to enable the cassette to be moved around in said housing (102) during the extraction process;

(e) a fluid conduit (not shown) for directing the solution into the sample cassette as and when such solution is required;

(f) a positive pump (112) for circulating solutions to the cassette; and

(h) a negative pump (114) to produce vacuum for the suction of solutions from the cassette during the extraction process.

The automated apparatus (100) as illustrated in Figure 2 is able to perform the extraction process automatically within the apparatus (100). The solutions required during the extraction process will be channeled from the solution dispensing apparatus (10) as attached thereto into the automated apparatus (100) when such solutions are required. By using the automated apparatus (100) of the present invention, an user can obtain the target biological material by only inserting the biological sample which is already placed in a cassette into the apparatus (100) via its input bay (104). The apparatus (100) is then programmed with the type of biological material to be extracted and the sample used. The apparatus will then carries out all the necessary steps until the target biological material is obtained and the cassette with the target biological materials will be ejected from its output bay (106) for collection.

The foregoing descriptions of specific embodiments of the present invention have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed, and obviously many modification and variations are possible. The embodiments were chosen and described in order to best explain the principles of the invention and its practical application, to hereby enable others skilled in the art to best utilize the invention and various embodiments with various modifications as are suited to the particular are contemplated. It is intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims

1. A method of extracting biological material from a biological sample via a sequence of procedures which involves the mixing of said biological sample with a series of solutions at their effective concentrations wherein said series of solutions is obtainable from a group of pre-determined chemicals which are able to be customized according to the requirements of the target biological material to be extracted and the biological sample from which the target biological material is extracted.

2. A method of extracting biological material from a biological sample according to Claim 1 wherein said group of pre-determined chemicals comprises of alkali metal chloride salt, detergent, concentrated weak organic carboxylic acid, polyethers, alkali metal acetate salt, chelating agent, alkali metal hydroxide and RNase inhibitor.

3. A method of extracting biological material from a biological sample according to Claim 2 wherein said alkali metal chloride salt is present in a concentration of about 1 - 5 M.

4. A method of extracting biological material from a biological sample according to Claim 3 wherein said alkali metal chloride salt is sodium chloride.

5. A method of extracting biological material from a biological sample according to Claim 2 wherein said detergent is present in a concentration of about

2 - 20% weight by volume (w/v).

6. A method of extracting biological material from a biological sample according to Claim 5 wherein said detergent is sodium dodecyl sulphate (SDS).

7. A method of extracting biological material from a biological sample according to Claim 2 wherein said concentrated weak organic carboxylic acid is present in a concentration of about 50 - 100% volume by volume (v/v).

8. A method of extracting biological material from a biological sample according to Claim 7 wherein said concentrated weak organic carboxylic acid is glacial acetic acid.

9. A method of extracting biological material from a biological sample according to Claim 2 wherein said polyethers is present in a concentration of about 20 - 60% weight by volume (w/v).

10. A method of extracting biological material from a biological sample according to Claim 9 wherein said polyethers is polyethylene glycol (PEG).

11. A method of extracting biological material from a biological sample according to Claim 2 wherein said alkali metal acetate is present in a concentration of about 1 - 5 M in a pH of about 4 - 6.

12. A method of extracting biological material from a biological sample according to Claim 11 wherein said alkali metal acetate salt is any one or combination of sodium acetate and potassium acetate.

13. A method of extracting biological material from a biological sample according to Claim 2 wherein said chelating agent is present in a concentration of about 0.2 - 3M.

14. A method of extracting biological material from a biological sample according to Claim 13 wherein said chelating agent is ethylenediaminetetra acetic acid (EDTA).

15. A method of extracting biological material from a biological sample according to Claim 2 wherein said alkali metal hydroxide is present in a concentration of about 1 - 5 M.

16. A method of extracting biological material from a biological sample according to Claim 15 wherein said alkali metal hydroxide is sodium hydroxide.

17. A method of extracting biological material from a biological sample according to Claim 2 wherein said RNase inhibitor is present in a concentration of about 10 - 100% by volume.

18. A method of extracting biological material from a biological sample according to Claim 17 wherein said RNase inhibitor is diethylpyrocarbonate (DEPC).

19. A method of extracting biological material from a biological sample according to Claim 1 wherein said sequence of procedures comprises the steps of: a), adding cellular disruption solution to said biological sample; b). incubating the disrupted sample from step a at a p re-determined temperature; c). adding separation solution to the incubated sample from step b to form a mixture; d). spinning the resulting mixture from step c in a pre-determined condition to obtain supernatant; e). adding extraction solution to the supernatant obtained from step d to form a mixture; f). spinning the resulting mixture from step e in a pre-determined condition to obtain the pellet which comprises the target biological material; wherein said cell disruption solution, separation solution and extraction solution are obtainable from the chemicals as claimed in Claims 2 - 18 and are able to be customized into different formulations according to the requirements of the target biological material to be extracted and the biological sample from which the target biological material is extracted.

20. A method of extracting biological material from a biological sample according to Claim 19 wherein said method further comprises a step of subjecting the biological sample that is in solid form to a lyses process prior to the step of adding the cell disruption solution to the sample.

21. A method of extracting biological material from a biological sample according to Claim 20 wherein said lyses process is performed by physical or chemical means at a low temperature to inhibit protein activity.

22. A method of extracting biological material from a biological sample according to Claims 20 to 21 wherein said lyses process is preferably performed by cutting, ultrasonication, homogenization or grinding of the solid samples into powder form in the presence of liquid nitrogen.

23. A method of extracting biological material from a₍ biological sample according to Claim 19 wherein said method further comprises the step of subjecting the biological sample to an enzymatic treatment if the sample comprises gram positive bacteria, yeast or cell culture prior to the step of adding the cell disruption solution to the sample.

24. A method of extracting biological material from a biological sample according to Claim 19 wherein said method further comprises a step of adding denaturing solution which comprises sodium hydroxide (NaOH) to the biological sample prior to the step of adding the cellular disruption solution to the sample for the extraction of plasmid DNA.

25. A method of extracting biological material from a biological sample according to Claim 19 wherein said method further comprises a step of adding RNase to the cellular disruption solution for the extraction of genomic or plasmid DNA.

26. A method of extracting biological material from a biological sample according to Claim 19 wherein said cellular disruption solution comprises chelating agent and detergent.

27. A method of extracting biological material from a biological sample according to Claim 19 wherein said cellular disruption solution comprises chelating agent and alkali metal chloride salt for the use in the extraction of native protein.

28. A method of extracting biological material from a biological sample according to Claims 26 to 27 wherein said chelating agent is ethylenediaminetetra acetic acid (EDTA), said detergent is sodium dodecyl sulphate (SDS) and said alkali metal hydroxide is sodium hydroxide (NaOH).

29. A method of extracting biological material from a biological sample according to Claim 19 wherein said incubation of step b is performed at about 37°C for about 5 - 10 minutes.

30. A method of extracting biological material from a biological sample according to Claim 19 wherein said separation solution comprises alkali metal acetate salt with the pH level adjusted to about 4 - 6 by using an appropriate volume of glacial acetic acid.

31. A method of extracting biological material from a biological sample according to Claim 30 wherein said alkali metal acetate salt is any one or combination of sodium acetate and potassium acetate.

32. A method of extracting biological material from a biological sample according to Claim 19 wherein said spinning of steps d and f is accomplished at about 10,000 - 15,00Og at room temperature for about 1 - 10 minutes.

33. A method of extracting biological material from a biological sample according to Claim 19 wherein said extraction solution comprises polyethers and alkali metal chloride salt.

34. A method of extracting biological material from a biological sample according to Claim 33 wherein said polyethers is polyethylene glycol (PEG) and said alkali metal chloride salt is sodium chloride.

35. A method of extracting biological material from a biological sample according to Claim 19 wherein the volume of said extraction solution used is substantially equivalent to that of the supernatant obtained from step d.

36. A method of extracting biological material from a biological sample according to Claim 19 wherein said method further comprises the step of mixing sterile distilled water or buffer with the dried pellet obtained from step e for storage purposes.

37. A kit for extraction of biological material according to any one of the preceding claims wherein said kit comprises a series of solutions obtainable from a group of chemicals as claimed in Claims 2 - 18.

38. A kit for extracting biological material according to any one of the preceding claims wherein said kit comprises at least of: a. cellular disruption solution obtainable from Claims 26 - 28 b. separation solution obtainable from Claims 30 - 31 c. extraction solution obtainable from Claims 33 - 34 wherein said cell disruption solution, separation solution and extraction solution are able to be customized according to the requirements of the extraction and are usable for a wide range of organisms to obtain high yield and purity.

39. A kit for extracting biological material according to claim 38 wherein said kit further comprises denaturing solution which comprises sodium hydroxide (NaOH) for the use in the extraction of plasmid DNA.

40. A kit for extracting biological material according to claim 38 wherein said kit further comprises RNase for the use in the extraction of genomic or plasmid DNA.

41. An apparatus for dispensing solutions required for the extraction of biological material wherein the concentrations and volumes of the solutions required can be pre-programmed or customized according to the requirements for the extraction of target biological material.

42. An automated apparatus to carry out the method according to Claims 1 - 36 wherein said apparatus comprises of:

(a) a computer controller (not shown) for controlling said automated apparatus

(100); (b) a liquid dispensing unit (10) for the dispensing of solutions;

(c) a housing (102) having:

(iii) at least a waste container (108) for collection of waste fluid from the extraction process;

(d) a mechanical moving part (110) for holding of the sample cassette and to enable the cassette to be moved around in said housing (102) during the extraction process;

(f) a positive pump (112) for circulating solutions to the cassette; and

(i) a negative pump (114) to produce vacuum for the suction of solutions from the cassette during the extraction process.