Description:
FIELD OF INVENTION
The present invention is to provide a method for extraction and isolation of plasmid DNA that enables obtaining extracted plasmid DNA in a short span of time.

BACKGROUND OF INVENTION
There are many existing methods for nucleic acids isolation and manipulation, the methods have one or more limitations. The limitations include requiring the use of hazardous chemicals, requiring extra drying steps, being difficult to automate, resulting in nucleic acids solution containing contaminants, causing DNA denaturation or not cost effective, etc. Some of the methods and limitations have been described in U.S. Pat. Nos. 5,523,231, 5,705,628 and 7,022,835 and WO/1984/001503.
As the method for extracting free DNA, TRizol method and centrifugal column method are generally used. The TRizol method is increasingly abandoned at present because of toxicity to operators and the centrifugal column method is difficult to realize complete automatic extraction. Also, it is not suitable for treatment of a large number of samples.
The advent of demanding molecular biology applications has increased the need for high-throughput, and preferably readily automatable purification protocols capable of producing high quality nucleic acid preparations. Although recent technological advancements and the advent of robotics have facilitated the automation of sequencing reactions and gel reading steps, throughput is still limited by the availability of readily automatable methods of nucleic acid purification.
Separation media, such as chromatography media and filtration media, are often associated with non-satisfactory properties to some end. Important factors in this field are e.g. the mass transport properties of the media, the flow properties thereof when used in chromatography columns or as membranes, cumbersome and non-reliable methods of preparation etc. Hence, there is an ongoing development to seek improvements in this field.
Recently, many products referred to as magnetic beads and a number of products for efficient handling of these beads have been presented. The magnetic beads are most

commonly used in combination with attached ligands having affinity for different substances. The most commonly encountered examples are metal chelating ligands (of IMAC type) intended for use in combination with His-tags and glutathione intended for use in combination with GST (Gluthathione S transferase). Other examples are a variety of different IgG's with different specificity.
U.S. Pat. No. 5,834,121 describes composite magnetic beads. Polymer coated metal oxide particles that are encapsulated in a rigid and solvent stable polymer of vinyl monomers in order to retain the metal oxide particles during harsh conditions. The primary beads are enclosed in a micro porous polymer bead which is capable of swelling in organic solvents and allowing for further functionalization in order to be useful for organic synthesis. This procedure is aiming for hydrophobic beads.
U.S. Pat. No. 6,204,033 describes preparation of polyvinyl alcohol-based magnetic beads for binding biomolecules. Preparation of magnetic beads by polyvinyl alcohol in water containing magnetic particles. The final beads contain hydroxyl functionalities that can be further derivatized in order to couple biomolecules. It is claimed that these magnetic beads can be grafted with vinyl monomers carrying various functional groups.
There is a need for simple and short method for isolation of DNA. The invention herein provides a quick and easy to perform method for isolation of plasmid DNA.

SUMMARY OF THE INVENTION
The present invention is a method of separating plasmid DNA from a solution containing plasmid DNA. The method comprises a first step of reversibly binding plasmid DNA non-specifically to solid surface materials. It is the objects of the present invention to provide method for purifying plasmid nucleic acids which at least partially avoid the disadvantages of the methods known in the prior art and which in particular enables a simple and cost-effective purification of a large number of samples. It is another object to provide methods for binding and releasing nucleic acids from the solid phase materials. It is yet another object to provide such reagent compositions for releasing bound nucleic acids from solid phase materials.

DETAILED DESCRIPTION OF THE INVENTION
The present invention is to provide a method for isolation of nucleic acids from a solution based on the principle that nucleic acids bind non-specifically and reversibly to solid phase surfaces with multi-amide-bond functional groups at certain salt and pH conditions. The method comprises contacting the nucleic-acids-containing solution with a solid phase material with functional groups which are able to bind the nucleic acids to the material and then extracting the nucleic acids. The solid phase materials can be in the form of particles, microparticles, fibers, beads, membranes, and other supports such as test tubes and microwells, pipette tips. As a result, methods for cost-effective, convenient and rapid separation of polynucleotides such as DNA, RNA and plasmid nucleic acid, from other molecules, such as proteins, lipids, polysaccharides, nucleotides are postulated. The following is a description of the present invention with reference to nucleic acids as exemplified by DNA. It is to be understood that the present invention, is also useful for separation of RNA in a similar manner.
One embodiment of the present invention is a method of separating plasmid DNA from a solution containing plasmid DNA. The method comprises a first step of reversibly binding plasmid DNA non-specifically to solid surface materials, such as magnetic particles whose surfaces are coated with functional groups with multiple amide bonds.
In one embodiment, the magnetic iron particles/ beads are combined with a solution of DNA with low ionic strength and a pH suitable for binding DNA to the solid surface of magnetic particles or beads. The salt concentration is from 0.1 to 100 mM; the pH is from 5 to 10. As a result, DNA is bound to the surfaces of the magnetic particles/ beads. Subsequently, the iron particles in the resulting combination are separated from the supernatant. The iron particles optionally, can be washed with a suitable wash buffer before they are contacted with a suitable elution buffer to elute and separate the DNA from the magnetic particles/ beads. In a final step, the magnetic particles are separated from the elution buffer, which contains the polynucleotides in solution. The magnetic particles are separated from elution buffer by, for example, filtration, centrifugation or applying a magnetic field.
Preferably, the releasing step is performed at a pH greater than 11 but less than 12 to maintain the integrity of the nucleic acids and to allow retrieval of nucleic acids. Salt, e.g., NaCl, KCl, at a concentration of less than 100 mM may be used to release nucleic acids at a pH lower than 11 at the elution step. Once the bound DNA has been eluted, the solid phase is separated from the elution buffer.
The present invention provides a method for extracting plasmid DNA of a sample by iron beads method, so that the yield of the extraction of the free nucleic acid/DNA is improved. The extraction process does not involve toxic reagents such as chloroform phenol and the like.

The invention provides a method for extracting free plasmid DNA of a sample, which comprises a lysate, a binding solution, a washing solution I, a washing solution II, an eluent and iron beads and magnetic shaft, wherein,
the lysing solution comprises of 100 - 500mM NaOH and 0.5 - 2% SDS;
the washing solution I comprises 1M Tris and 70% ethanol;
the washing solution II comprises 75% ethanol;
the binding solution comprises guanidine isothiocyanate and isopropanol;
and the elution buffer comprises 1M Tris;

As a preferred embodiment, the washing liquid I or solution I comprises the following components in proportion: 0.3-0.5% of 1M/L Tris-HCl and 60-80% absolute ethyl alcohol;
the washing liquid II or solution II comprises the following components in percentage by weight: 75%, ethanol and the elution buffer comprises 1M Tris.

The lysing solution may contain SDS and Buffer, wherein the SDS is an anionic detergent and the proteins and proteinase K (cut serine protein) are combined to form SDS/protein/polysaccharide complexes to release nucleic acids. When the lysate is used for lysing a plasma sample, the rapid and thorough separation of nucleic acid and nucleic acid binding protein is ensured, a large amount of nucleic acid is released, and the nucleic acid released from the lysate is efficiently and specifically adsorbed on iron beads coated with silica.

The iron bead solution is a solution of silica coated nano iron beads. The iron beads have a magnetic core and the outside is provided with a coating layer, the surface of the iron bead is distributed with a plurality of active groups, the magnetic bead can be coupled with biochemical reagents such as cells, proteins, nucleic acid, enzymes and the like on a microscopic interface, and the iron beads can move directionally under the action of an external magnetic field to realize separation from a medium solution. Under the action of the externally applied magnetic field, the iron beads and the nucleic acid molecules are specifically identified and efficiently separated and DNA in biological samples, environmental samples and the like can be separated.
The magnetic microparticles/beads used in the method of the present invention comprise a magnetic metal oxide core which is generally surrounded by an adsorptively or covalently bound silica coat to which a wide variety of bioaffinity adsorbents can be covalently bound.

The method is simple and involves transferring the samples to be extracted containing target DNA fragments, a restriction enzyme cutting system or other enzymatic reaction liquid block into a centrifuge tube, pipetting and uniformly mixing, and allowing DNA to be adsorbed on iron beads; impurities are removed by two washes and finally purified DNA is eluted with a small amount of eluent or water.

The method greatly improves the extraction yield. The notable advantage of the method is that no toxic reagents such as chloroform and phenol are used for extraction.

As an embodiment the invention provides a kit for extracting plasmid DNA of a sample that comprises a lysate, a washing solution I, a washing solution II, a binding solution, an eluent and magnetic beads. The kit further comprises a pre-dispensed reagent plate and a magnetic shaft.

The invention also provides a method for extracting the free nucleic acid/DNA of the plasma/blood sample.

EXAMPLE 1
The extraction of plasmid DNA was carried out by extracting bacterial cells. The recombinant E.coli culture was grown overnight (14-16 hours old culture) in a medium containing appropriate antibiotic, for instance ampicillin or kanamycin etc. The appropriate volume of the culture (0.5 to 1.5 ml) was taken into a 2.0 ml capped microcentrifuge tube and the cells were centrifuged at =12,000 x g [˜13,000 rpm] for 1 minute. The supernatant culture medium was discarded. For good plasmid DNA yield, the density of the culture should be around 3.0 x 10 6 cells/ml. The optimal volume of culture to be used is calculated by dividing the cell mass with the O.D600 value.

The bacterial pellet is suspended in 250 µl of Resuspension Solution with composition 50 - 200 mM Tris-Cl, 10mM EDTA, pH 8.0. The appropriate amount of RNase A Solution (8 µl to 18 µl) is added to resuspended pellet solution and mixed well by gentle pipetting till no cell clumps are visible. It is very important that homogenous suspension is obtained as incomplete resuspension results in poor recovery.

The Lysis Solution approx.150 -400µl was added to the suspended pellet to lyse the cells. It is mixed thoroughly by gently inverting the tube 4-6 times.

The Neutralization Solution (approx. 200-450 µl) was added and immediately mixed thoroughly by gently inverting the tube 4-6 times. The neutralization solution comprise 20 -30% Guanidine hydrochloride, 1-5% Acetic acid. The solution turns cloudy. The solution was centrifuged at approximately 12,000 x g (˜13,000 rpm) for 10 minutes to obtain a compact white pellet. A compact white pellet was formed. For obtaining clear supernatant, the solution was transferred to a fresh tube and spined for an additional minute at 12,000 x g (˜13,000 rpm) to remove the interfering salts/precipitates completely. The supernatant obtained is a pre-processed sample.

EXAMPLE 2
The extraction of plasmid DNA was carried out from the pre-processed sample.
The iron beads (20ul) are added to the pre-processed sample (700ul) and are allowed to bind for about 10 to 20 minutes, preferably 15 minutes. The magnetic shaft is placed in the pre-processed sample. The iron beads adsorbed with the plasmid DNA get attracted towards the magnetic shaft. The beads on the shaft are washed in washing buffer I and then with washing buffer II. The iron beads are then transferred to elution buffer. The plasmid DNA is eluted in the elution buffer and the eluate contains pure plasmid DNA. The process of extraction of plasmid DNA from the pre-processed sample can be performed manually or can be automated. The automated process can purify plasmid DNA from more than one sample, particularly up to 96 samples at a time.

We Claim

1) A method of isolation of plasmid DNA comprising the steps of:
a] lysing and pre-processing of the recombinant bacterial culture from which plasmid is to be extracted,
b] mixing the pre-processed suspension with silica coated iron beads,
c] separating the iron beads on which plasmid DNA is adsorbed,
d] washing the plasmid DNA adsorbed iron beads and
e] eluting the plasmid DNA from the iron beads.

2) The method of isolation of plasmid DNA as claimed in claim 1 wherein exponential phase recombinant bacterial cells are suspended in resuspension solution having composition as 50 - 200 mM Tris-Cl, 10mM EDTA, pH 8.0.

3) The method of isolation of plasmid DNA as claimed in claim 1 wherein the cells suspended in resuspension solution are lysed with lysis solution having composition as 100 - 500mM NaOH and 0.5 - 2% SDS.

4) The method of isolation of plasmid DNA as claimed in claim 1 wherein the lysed cells are neutralized with neutralization solution having composition as 20 -30% guanidine hydrochloride, 1 - 5% acetic acid and centrifuged to obtain clear supernatant pre-processed solution.

5) The method of isolation of plasmid DNA as claimed in claim 1 wherein the plasmid DNA adsorbed iron particles are separated by a magnetic shaft.

6) The method of isolation of plasmid DNA as claimed in claim 1 wherein the plasmid DNA adsorbed iron particles are washed twice with first washing solution I with composition 0.3-0.5% of 1M/L Tris-HCl and 60-80% absolute ethyl alcohol and second washing solution II with composition 0.3-0.5% of 1M/L Tris-HCl and 60-80% absolute ethyl alcohol respectively.

7) The method of isolation of plasmid DNA as claimed in claim 1 wherein the washed iron particles with adsorbed plasmid DNA are eluted in the elution buffer with composition of 1M Tris.

8) The method of isolation of plasmid DNA as claimed in claim 1 wherein the eluate with pure plasmid DNA is separated by centrifugation.

9) The method of isolation of plasmid DNA as claimed in any of claims 1 to 6 wherein the method is automated to process more than one sample at a time.
, Claims:We Claim

1) A method of isolation of plasmid DNA comprising the steps of:
a] lysing and pre-processing of the recombinant bacterial culture from which plasmid is to be extracted,
b] mixing the pre-processed suspension with silica coated iron beads,
c] separating the iron beads on which plasmid DNA is adsorbed,
d] washing the plasmid DNA adsorbed iron beads and
e] eluting the plasmid DNA from the iron beads.

2) The method of isolation of plasmid DNA as claimed in claim 1 wherein exponential phase recombinant bacterial cells are suspended in resuspension solution having composition as 50 - 200 mM Tris-Cl, 10mM EDTA, pH 8.0.

3) The method of isolation of plasmid DNA as claimed in claim 1 wherein the cells suspended in resuspension solution are lysed with lysis solution having composition as 100 - 500mM NaOH and 0.5 - 2% SDS.

4) The method of isolation of plasmid DNA as claimed in claim 1 wherein the lysed cells are neutralized with neutralization solution having composition as 20 -30% guanidine hydrochloride, 1 - 5% acetic acid and centrifuged to obtain clear supernatant pre-processed solution.

5) The method of isolation of plasmid DNA as claimed in claim 1 wherein the plasmid DNA adsorbed iron particles are separated by a magnetic shaft.

6) The method of isolation of plasmid DNA as claimed in claim 1 wherein the plasmid DNA adsorbed iron particles are washed twice with first washing solution I with composition 0.3-0.5% of 1M/L Tris-HCl and 60-80% absolute ethyl alcohol and second washing solution II with composition 0.3-0.5% of 1M/L Tris-HCl and 60-80% absolute ethyl alcohol respectively.

7) The method of isolation of plasmid DNA as claimed in claim 1 wherein the washed iron particles with adsorbed plasmid DNA are eluted in the elution buffer with composition of 1M Tris.

8) The method of isolation of plasmid DNA as claimed in claim 1 wherein the eluate with pure plasmid DNA is separated by centrifugation.

9) The method of isolation of plasmid DNA as claimed in any of claims 1 to 6 wherein the method is automated to process more than one sample at a time.