GB2455780A - Nucleic acid separation - Google Patents

Nucleic acid separation Download PDF

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GB2455780A
GB2455780A GB0724976A GB0724976A GB2455780A GB 2455780 A GB2455780 A GB 2455780A GB 0724976 A GB0724976 A GB 0724976A GB 0724976 A GB0724976 A GB 0724976A GB 2455780 A GB2455780 A GB 2455780A
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nucleic acid
magnetic particle
solution
polyethylene glycol
magnetic
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Zainulabedin Mohamedali Saiyed
Nanappan Ramchand Chaniyilparampu
Ajay Kapoor
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/002—High gradient magnetic separation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/005—Pretreatment specially adapted for magnetic separation
    • B03C1/01—Pretreatment specially adapted for magnetic separation by addition of magnetic adjuvants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/0098—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation

Abstract

The present invention provides a method of reversibly binding a nucleic acid molecule to a non-coated magnetic particle. The method comprises combining non-coated magnetic particles with a solution containing at least one nucleic acid molecule. An adsorption buffer is provided to the solution in a concentration sufficient to bind the nucleic acid to the surface of magnetic particle, wherein the adsorption buffer comprises a polyethylene glycol and a monovalent salt, thereby binding the nucleic acid to the magnetic particle. Also mentioned is the method comprising the following further steps: separating the magnetic particles having nucleic acid bound to the surface and eluting the bound nucleic acid from the magnetic particles.

Description

Nucleic acid separation The present invention relates to a method for reversibly binding nucleic acid molecules.
The invention also relates to a method of isolating nucleic acid molecules, particularly genomic DNA, from a sample.
BACKGROUND
Isolation of DNA is a prerequisite step for many molecular biology techniques. The separation of DNA from the complex mixtures in which they are often found is frequently necessary before other studies and procedure like sequencing amplification, hybridization, detection etc. Conventionally, extracting DNA involves cell lysis followed by removal of contaminating cellular components such as proteins, lipids and carbohydrates; and finally isolating DNA using a series of precipitation and centrifugation steps, which are difficult to automate.
More recently extraction methods have been proposed that employ a mobile solid phase.
Protocols for isolating nucleic acid molecules are described in US 6,821,757, which discloses a method for separating and isolating circular nucleic acids from a mixture having different species of nucleic acids other than circular nucleic acids wherein the mixture is treated under alkaline conditions at a pH>8 with a solid matrix consisting * ** essentially of a silica material in the presence of at least one chaotropic substance. * I S * I. I... * I
Protocols for isolating nucleic acid molecules are described in US 5,705,628, which describes a method for separating polynucleotides, from a solution containing polynucleotides, by reversibly and non-specifically binding the polynucleotides to a solid surface such as a magnetic microparticle having a functional group-coated surface. In particular the document discloses methods of reversibly binding DNA to magnetic I...
:.30 particles having a carboxyl group-coated surface.
US 5,091,206 discloses magnetically responsive polymer particles comprising polymeric core particles coated evenly with a layer of polymer containing magnetically responsive metal oxide. A wide variety of polymeric particles with sizes ranging from 1 to 100 microns can be used as core particles and transformed into magnetically responsive polymer particles. The surface of these magnetically responsive polymer particles can be coated further with another layer of functionàlized polymer. These magnetically responsive polymer particles can be used for passive or covalent coupling of biological material such as antigens, antibodies, enzymes of DNA/RNA hybridization and used as a solid phase for various types of immunoassays, DNA/RNA hybridization probes assays, affinity purification, cell separation and other medical, diagnostic and industrial applications.
Application of magnetite and silica-magnetite composites to the isolation of genomic DNA (J. Chromatogr A 2000; 890: 159-166) discloses isolation of genomic DNA from maize kernels using magnetite and silica-magnetite..
WO 96/18731 by Deggerdal Arne et al discloses method of isolating nucleic acid from a sample by contacting said sample with a detergent and a solid support, whereby soluble nucleic acid in said sample is bound to the support, and separating said support with bound nucleic acid from the sample.
US 6368800 discloses isolation of biological target materials, particularly nucleic acids, such as DNA and RNA, from other substances in a medium using silica magnetic particles. The method involves forming a complex of the silica magnetic particles and the biological target material in a mixture of the medium and particles, separating the complex from the mixture using external magnetic force, and eluting the biological target material from the complex. * ** *. *
Isolation of plasmid DNA using magnetite as a solid phase adsorbent (Analytical Biochemistry 1998; 262: 92-94) discloses a method which includes binding of plasmid DNA to the solid support (magnetite -Fe304) in the presence of adsorption buffer.
Known protocols for nucleic acid isolation which involve the use of coated magnetic S...
particles or beads, in particular silica coated particles or beads, are both time consuming *. 30 and costly as coating reduces the surface area available for binding, thereby reducing the yield of nucleic acid isolated by each particle.
There remains a need for a method for isolating biological entities, particularly genomic nucleic acids, using a magnetically responsive particle capable of rapidly and efficiently directly isolating such entities sufficiently free of contaminants to be used in molecular biology procedures.
The present invention provides a novel technique for isolation of genomic DNA using magnetic nanoparticles as mobile solid phase. The present invention describes isolation of genomic DNA directly from crude cell lysate without the requirement for the initial lysis step followed by processing of the sample and finally separating out the cleared cell lysate by centrifugation.
The yield of genomic DNA obtained using the technique of the present invention, is either higher or equivalent to that reported earlier when silica support was used as an adsorbent. Also, the yields of isolated DNA were substantially higher in comparison with a commercially available kit.
BRIEF SUMMARY OF THE DISCLOSURE
In a first aspect the invention provides a method of reversibly binding a genomic DNA molecule to a non-coated magnetic particle comprising: a) combining at least one non-coated magnetic particle with a solution containing at least one nucleic acid molecule; b) providing an adsorption buffer to the solution in a concentration sufficient to bind the DNA molecule to the surface of the at least one magnetic particle, wherein the adsorption buffer comprises PEG6000 and NaCI, thereby binding the nucleic acid to the magnetic particle. * ** * * S
In a further aspect, the invention provides a method of reversibly binding a nucleic acid molecule to a non-coated magnetic particle comprising: a) combining at least one non-coated magnetic particle with a solution containing at least one nucleic acid molecule; b) providing an adsorption buffer to the solution in a concentration *.S* sufficient to bind the nucleic acid molecule to the surface of the magnetic particles, wherein the adsorption buffer comprises a polyalkylene glycol and a monovalent salt, thereby binding the nucleic acid molecule to the magnetic particle.
In a preferred embodiment the non-coated magnetic particle possess hydroxyl groups on the surface thereof.
In a preferred embodiment the nucleic acid molecule is a genomic DNA molecule.
In a preferred embodiment the polyalkylene glycol is polyethylene glycol (PEG).
In a preferred embodiment the polyethylene glycol has a molecular weight between 4000 to 10,000. More preferably, the polyethylene glycol has a molecular weight of 6000.
In a preferred embodiment, the adsorption buffer comprises from 0 to 40% volume PEG, preferably 10 to 30% volume PEG, more preferably 15 to 25% volume PEG.
In a preferred embodiment the monovalent salt is a sodium salt. More preferably the salt is NaCI.
In a preferred embodiment, the adsorption buffer comprises 1 to 10 M of monovalent salt, preferably 1 to 5 M of monovalent salt, more preferably 1 to 5 M NaCI.
In a preferred embodiment the polyethylene glycol is PEG6000 and the monovalent salt is NaCl.
In a preferred embodiment the non-coated magnetic particle possess hydroxyl groups on the surface thereof.
In a preferred embodiment the invention the magnetic particles comprise a metal oxide.
*::: Preferably, the metal oxide is a divalent or trivalent iron oxide, more preferably Fe304. *.
In a preferred embodiment the invention the magnetic particles are nanoparticles.
Preferably, the nanoparticles have a mean diameter range from 5 to 100 nanometers.
In a further aspect, the invention provides a method of separating nucleic acid molecules *.SS : from a solution containing at least one nucleic acid molecule, comprising: *. 30 a) combining at least one non-coated magnetic particle with the solution; b) providing an adsorption buffer to the solution in a concentration sufficient to bind the nucleic acid molecule to the surface of the magnetic particle, wherein the adsorption buffer comprises a polyethylene glycol and a monovalent salt; c) separating the at least one magnetic particle having nucleic acid molecules bound to the surface thereof from the solution; and d) eluting the bound nucleic acid molecules from the magnetic particles, thereby separating nucleic acids molecules from a solution containing at least one nucleic acid molecule.
In a preferred embodiment the non-coated magnetic particle possess hydroxyl groups on the surface thereof.
In a preferred embodiment the nucleic acid molecule is a genomic DNA molecule.
In a preferred embodiment the polyalkylene glycol is polyethylene glycol (PEG).
In a preferred embodiment the polyethylene glycol has a molecular weight between 4000 to 10,000. More preferably, the polyethylene glycol has a molecular weight of 6000.
In a preferred embodiment, the adsorption buffer comprises from 0 to 40% volume PEG, preferably 10 to 30% volume PEG, more preferably 15 to 25% volume PEG.
In a preferred embodiment the monovalent salt is a sodium salt. More preferably the salt is NaCI.
In a preferred embodiment, the adsorption buffer comprises 1 to 10 M of monovalent *:*::* salt, preferably 1 to 5 M of monovalent salt, more preferably 1 to 5 M NaCl. * ***
In a preferred embodiment the polyethylene glycol is PEG6000 and the monovalent salt is NaCI.
In a preferred embodiment the non-coated magnetic particle possess hydroxyl groups on the surface thereof.
S
In a preferred embodiment of the invention the magnetic particles comprise a metal oxide. Preferably, the metal oxide is a divalent or trivalent iron oxide, more preferably Fe304.
In a preferred embodiment of the invention the magnetic particles are nanoparticles.
Preferably, the nanoparticles have a mean diameter range from 5 to 100 nanometers.
In a preferred embodiment the separation of the magnetic particles from the solution is
achieved by the application of a magnetic field.
In a preferred embodiment eluting the bound nucleic acid from the magnetic particle is achieved by contacting the magnetic particles having nucleic acid bound to the surface thereof with an elution buffer which separates the nucleic acid from the magnetic particle. Preferably, the elution buffer is TE elution buffer. Alternatively, the elution buffer is pure water..
In a preferred embodiment, the invention further comprises the step of precipitating protein from the solution.
In a preferred embodiment of the invention proteins are precipitated from the solution by altering the dielectric constant of the solution.
Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.
Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. S* S.
* Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be . understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.
DETAILED DESCRIPTION
The present invention provides a method for the isolation of genomic DNA using magnetic nanoparticles as a mobile solid phase. In particular, the present invention provides for the isolation of genomic DNA directly from a cell sample, thereby removing the requirement to process the sample in order to obtain a sample free of protein and other nucleic acid molecules by centrifugation and phenol extraction steps. The method allows for the isolation of genomic DNA without the requirement for RNase treatment of the sample.
For example, genomic DNA isolation from whole blood, buffy coat, PBMCs (Peripheral blood mononuclear cells), cell cultures, tissue homogenate (rat brain, liver and heart), gram positive bacteria (Streptomyces flaviscieroticus), gram-negative bacteria (E. coil and S. typhil), early eukaryotes (S. cerevisiae and Dictyostelium discoideum) have been be optimized using the technique described in the present invention. Additionally, extraction of DNA fragments from agarose gel has been optimized using magnetic nanoparticles as solid phase support The solution comprising at least one nucleic acid molecule can be provided by cell lysis.
The yield of genomic DNA obtained using the technique of the present invention, is either higher or equivalent to that obtained when a silica support is used as. Moreover, the yields of isolated DNA were substantially higher in comparison with a commercially available kit.
Magnetic particles designed for use in nucleic acid isolation generally fall into either of two categories, those designed to reversibly bind nucleic acid materials directly, and *:*::* those designed to do so through at least one intermediary substance. The present invention employs magnetic particles designed to reversibly bind nucleic acid materials directly, i.e. a non-coated magnetic particle.
* Preferably, the particles are magnetic nanoparticles. Preferably, the mean diameter of the magnetic nanoparticles is in the range of 1-100 nanometers, 5 -100 nanometers, 5-nanometers or 5-20 nanometers, which provides a greater surface area on weight basis for DNA binding. Preferably the particles have a mean diameter of 40 nanometers Preferably the method of the invention is used to isolate genomic DNA, i.e. the DNA constituting the genome of a cell or organism, for example chromosomal DNA.
Alternatively, the method may be used to isolate other nucleic acid molecules, such as DNA, PNA or plasm ids.
The adsorption buffer of the invention provides the necessary conditions for the selective binding of the nucleic acid molecule, i.e. the selective binding of a genomic DNA molecule to the magnetic particle.
In a preferred embodiment the adsorption buffer comprises sodium chloride, a small molecule with high ionization potential. The adsorption buffer must also comprise a polyalkylene glycol, preferably polyethylene glycol (PEG), more preferably PEG-6000.
The inventors have found that when an adsorption buffer is employed in the present methods that comprises NaCl and PEG6000, the dielectric constant of the solution containing nucleic acid molecules, i.e. genomic DNA, is altered such that genomic DNA, but not proteins and other biomolecules such as RNA and cell debris, selectively binds to the magnetic particles.
In one embodiment, magnetic particles (Fe304) are added to cell lysate followed by addition of adsorption buffer (4 M sodium chloride (NaCI) and 20 % PEG-6000). After incubating the mixture for 5 mm, the magnetic particles bound to the DNA are separated from the rest of the material by application of a magnetic field. The magnetic pellet is washed twice in 50 % ethanol, followed by drying the pellet for few minutes. The pellet is finally suspended in sterile water or TE (Tris-EDTA, pH 7.8) buffer and the magnetic particles bound DNA is eluted by constant shaking and incubation at 65°C for 5 mm. The supernatant containing the eluted DNA is separated from the particles by application of
an external magnetic field. S...
The samples from which DNA isolation is optimized using this method include, but are not limited to, biological samples, such as whole blood, buffy coat, PBMCs (Peripheral blood mononuclear cells), cell cultures, tissue homogenate (rat brain, liver and heart), *...
: gram positive (Streptomyces flaviscieroticus), gram-negative bacteria (E. co/i and S. * 30 lyphil), early eukaryotes (S. cerevisiae and Dictyostelium discoideum), or samples such as agarose gels. The extraction of DNA fragments from agarose gel has been optimized using magnetic nanoparticles as solid phase support.
In accordance with the present invention, elution of DNA from agarose gel has also been performed using this method. The yield of genomic DNA obtained using this method was either higher or equivalent to that reported earlier when silica support was used as adsorbent.
Comparison of this method with a commercially available kit indicated that the yield of isolated DNA was 1.3 fold higher.
The present method involves isolation of genomic DNA directly from the crude sample; in contrast with the prior art where the process involves the initial lysis step followed by processing of the sample and finally separating out the cell lysate by centrifugation. The improved process of direct isolation with higher yield is not only technologically superior but also is of economic significance. The cell lysate is further used for isolation of DNA using magnetite or silica-magnetite.
The magnetic nanoparticles used in the method of the present invention are metal oxide preferably iron oxide which displays an amphoteric hydroxyl (-OH) group on the surface.
This -OH group can be used for linking variety of biomolecules through covalent coupling chemistries or under specific condition to tightly adsorb biomolecules such as genomic DNA.
In the present invention, use of magnetic nanoparticles offers several advantages; the nano-size of the particles provides a higher surface area (on a weight basis) for binding of the biomolecules; the particles of the present invention are uncoated (naked), which makes them highly susceptible to the external magnetic field; and due to the nano-size, *... the particles can exist as stable colloidal suspension that will not aggregate, allowing for S...
.,. 25 uniform distribution in a reaction mixture. *
* The magnetic nanoparticles were prepared in the laboratory by co-precipitating di and trivalent iron (Fe) ions by alkaline solution and treating under hydrothermal conditions.
.. The particles were stored in TE buffer (10 mM Tris -HCI and 1mM EDTA, pH 8.0) at a S..
* 30 suspension concentration of 20 mg/mI. All agarose gels were run on 0.8% final agarose .5 in lx TAE buffer. The field strength was 8V/cm with run times of about 60 mm. The gels were stained with ethidium bromide and visualized using a UV transilluminator.
The DNA extraction method of the present invention was tested for its efficiency and ease of use compared with a ommerciaJly available kit (QlAamp DNA Blood Mini Kit for blood and QlAamp DNA Mini Kit for cultured cells, Qiagen�).
The yield of DNA extracted using the method of the present invention was on average 1.3-fold greater than that using the commercially available method. Moreover, the method of the present invention can be carried out in a single microcentrifuge tube per sample, whereas the commercially available procedure requires a number of tube transfers.
The higher yield of genomic DNA obtained using magnetic nanoparticles as solid-phase support may be attributed to the nano-size of the particles, which provides increased surface area for binding of DNA and creation of optimal conditions in the presence of adsorption buffer (4M NaCI and 20% PEG 6000).
Additionally, the DNA from all the extractions was found to function satisfactorily in restriction endonuclease digestion. This indicates absence of any enzyme inhibitors in the extracted DNA.
Furthermore, the efficiency of the extracted DNA was also checked in PCR amplification.
A 226-bp fragment of GAPDH gene was successfully amplified using the genomic DNA extracted from whole blood as a template.
Following selected examples illustrates the applicability of the present invention, which are not limiting in any way.
Examples: SI.. * . S...
Example 1: Genomic DNA isolation from whole blood using magnetic nanoparticles as a solid phase support: 1. 30 p.1 of sample (whole blood), 30 p.1 of 1 % (w/v) detergent (SDS) solution were added.
2. The tube was mixed by gentle inversion for two or three times and incubated at 30 room temperaturefor2min.
3. After incubation, 10 p.1 of magnetic nanoparticles (20 mg/mI) were added to the cell lysate, followed by addition of 75 p.1 of adsorption buffer (4 M sodium chloride and 20% PEG 6000).
4. The suspension was mixed by inversion and allowed to stand at room temperature for at least 3 mm.
5. The magnetic pellet was immobilized by application of an external magnet and supernatant was removed.
6. The magnetic pellet was washed with 50 % ethanol and dried.
7. The pellet was then completely resuspended in 50 pi of TE buffer (pH 8.0) by repeated pipetting strokes and magnetic particle bound DNA was eluted by incubation at 65 °C for 5 mm with continuous agitation.
8. The magnetite particles were then immobilized with a magnet and the clear supernatant containing DNA was transferred to a fresh tube. The eluted DNA was analyzed using agarose gel electrophoresis.
The yields of recovered genomic DNA were up to 1.21.tg per 301.1.1 of whole blood.
The above procedure was also found to be applicable for genornic DNA isolation from buffy coat and peripheral blood mononuclear cells (PBMCs). However, in those cases only 10 tl of starting material was taken instead of 30 1.tl used for blood. This is due to the fact that buffy coat and PBMCs contains only nucleated cells Example 2: Genomic DNA isolation from cultured cells using magnetic nanoparticles as a solid phase support: Cultured cells used in the present invention were of colon carcinoma cell lines (HCT1 16).
For genomic DNA isolation, the cells were trypsinized and adjusted to a cell density of 7 x 106 cells per ml with PBS (phosphate buffered saline, pH 7.4).
1. 30 gI of cell suspension, which corresponds to 2 x i05 cells were taken in a * .* *****. microfuge tube, followed by addition of 30.tl of detergent (1% w/v SDS).
2. Same steps as blood DNA isolation (step 2 -8) were followed.
I
* * Example 3: Genomic DNA isolation from yeast cells (S. cereviceae) using magnetic nanoparticles as a solid phase support: The genomic DNA from yeast cells was purified by creating a cleared lysate, reversibly binding the genomic DNA to the magnetic nanoparticles, separating the magnetic 30 nanoparticles from rest of the components and finally eluting the bound DNA. The procedure used is described below: 1. S. cereviceae were grown in yeast potato dextrose (YPD) medium to a cell density of 108 cells per ml.
2. Preparation of a cleared cell lysate: Centrifuge to pellet the cells.
Supernatant was discarded and resupend the pellet in 100 uI sorbitol buffer containing zymolase (200U/ml), Incubate at 37°C for 10 mm.
Treat the cell suspension with 12.5 j.tl of detergent (10 % SDS) pIus 10 j.d of proteinase K (20 mg/mI) and incubate at 65°C for 10 mm.
Centrifuge and transfer supernatant to a fresh tube.
3. To the cleared cell lysate, 75 l of magnetic nanoparticles (10 mg/mI) were added followed by 200.il of adsorption buffer (4 M sodium chloride and 20% PEG 6000).
4. After the incubation at room temperature for 5 mm, the magnetic pellet was washed twice with 50% ethanol.
5. Finally, the pellet was resuspended in 50 j.tI of TE buffer and DNA eluted.
Example 4: Extraction of DNA from agarose gel using magnetic nanoparticles as a solid-phase support: The present invention was also found to be applicable for extraction of DNA fragments from agarose gels: In an optimized procedure, 1. Run DNA sample (23-kb) on a 0.8% agarose gel.
2. Visualize the DNA band using a UV transilluminator 3. Excise the band of interest with a sterile blade and transferred to a microcentrifuge tube.
* ** 4. Add 4 volumes of SSC (0.75 M sodium chloride, 0.0075 M sodium citrate, pH 7.0) buffer to the agarose plug and incubate at 80°C for 5 mm to allow agarose to melt.
5. After incubation, add 20.tl of magnetic nanoparticles (10 mg/mI), followed by addition of 200 pi of adsorption buffer (4M sodium chloride and 20% PEG 6000).
6. Mix the suspension by inversion and allowed it to stand at room temperature for 5 *.SU *..: mm.
*. 30 7. Immobilize the magnetic pellet by application of an external magnetic field and discard the supernatant.
8. Wash the magnetic pellet twice with 50 % ethanol.
9. Resuspend the pellet in 30 j.tl of TE buffer and elute DNA from magnetic nanoparticle by incubation at 65 °C with continuous agitation.
10. Finally, separate the particles magnetically and transfer the supernatant containing DNA to a fresh tube.
The yield of recovered DNA from agarose gel with the method described in the present invention was �=80%, whereas it was only 50 to 60% with conventional phenol extraction and glass wool spin column procedures.
The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.
All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.
Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.
The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed *.. in this specification (including any accompanying claims, abstract and drawings), or to *,25 any novel one, or any novel combination, of the steps of any method or process so *...
disclosed.
S 55*5
S S..

Claims (28)

1. A method of reversibly binding a genomic DNA molecule to a non-coated magnetic particle comprising: a) combining at least one non-coated magnetic particle with a solution containing at least one nucleic acid molecule; b) providing an adsorption buffer to the solution in a concentration sufficient to bind the at least one genomic DNA molecule to the surface of the at least one magnetic particle, wherein the adsorption buffer comprises PEG600 and NaCI, thereby binding the nucleic acid to the magnetic particle.
2. A method of reversibly binding a genomic DNA molecule to a non-coated magnetic particle comprising: a) combining at least one non-coated magnetic particle with a solution containing at least one nucleic acid molecule; b) providing an adsorption buffer to the solution in a concentration sufficient to bind the at least one DNA molecule to the surface of the at least one magnetic particle, wherein the adsorption buffer comprises a polyethylene glycol and a monovalent salt, thereby binding the nucleic acid to the magnetic particle.
3. A method according to claim 2, wherein the polyethylene glycol has a molecular weight between 4000 to 10,000. * ** * a a * **
25
4. A method according to claim 3, wherein the polyethylene glycol has a molecular weight of 6000.
*.SS.S * S
5. A method according to any one of claims 2 to 4, wherein the monovalent salt is a sodium salt. S..
6. A method according to claim 5, wherein the salt is NaCI.
7. A method according to claim 2, wherein the polyethylene glycol is PEG6000 and the monovalent salt is NaCI.
8. A method according to any preceding claim, wherein the non-coated magnetic particle comprises hydroxyl groups on the surface thereof.
9. A method of separating nucleic acid molecules from a solution containing at least one nucleic acid molecule, comprising; a) combining at least one non-coated magnetic particle with the solution; b) providing an adsorption buffer to the solution in a concentration sufficient to bind the nucleic acid molecule to the surface of the magnetic particle, wherein the adsorption buffer comprises a polyethylene glycol and a monovalent salt; c) separating the magnetic particles having nucleic acid bound to the surface thereof from the solution; and d) eluting the bound nucleic acid from the magnetic particles, thereby separating nucleic acids from a solution containing at least one nucleic acid molecule.
10. A method according to claim 9, wherein the nucleic acid molecule is a genomic nucleic acid molecule.
11. A method according to claim 9 or claim 10, wherein the polyethylene glycol has a molecular weight between 4000 to 10,000.
12. A method according to claim 11, wherein the polyethylene glycol has a molecular * Se weight of 6000. S...
*....S 25
13. A method according to any preceding claim, wherein the monovalent salt is a
SSSI
* sodium salt.
14. A method according to claim 13, wherein the salt is NaCI.
*** 30 S..
15. A method according to claim 9 or claim 10, wherein the polyethylene glycol is PEG6000 and the monovalent salt is NaCI.
16. A method according to any one of claims 9 to 15, wherein the non-coated magnetic particle comprises hydroxyl groups on the surface thereof.
17. A method according to any one of claims 9 to 16, wherein separation of the magnetic particles from the solution is achieved by the application of a magnetic field.
18. A method according to any one of claims 9 to 16, wherein the eluting the bound nucleic acid from the magnetic particle is achieved by contacting the magnetic particles having nucleic acid bound to the surface thereof with an elution buffer which separates the nucleic acid from the magnetic particle.
19. A method according to claim 18, wherein the elution buffer is TE elution buffer.
20. A method according to claim 18, wherein the elution buffer is pure water.
21. A method according to claim 18, wherein the elution buffer is a selective elution buffer.
22. A method according to claim 19, wherein the selective elution buffer is a size selective elution buffer.
21. A method according to any one of claims 9 to 21, further comprising the step of precipitating protein from the soiution.
22. A method according to claim 21, wherein proteins are precipitated from the solution by altering the dielectric constant of the solution. * ** * S S * S.
23. A method according to any one of claims 1 to 22, wherein the magnetic particles comprise a metal oxide.
SeS*SS * .
24. A method according to claim 23, wherein the metal oxide is a divalent or trivalent iron oxide. *5*
25. A method according to claim 24, wherein the metal oxide is Fe304.
26. A method according to any one of claims 1 to 25, wherein the magnetic particles are nanoparticles.
27. A method according to claim 26, wherein the nanoparticles range from 5 to 100 nanometers in size.
28. A method according to any one of claims 9 to 27, wherein the non-coated magnetic particle comprises hydroxyl groups on the surface thereof. * S. * * S * .. **. * * *.. *
*.**S. * S
S
S *S.
S
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Cited By (1)

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Publication number Priority date Publication date Assignee Title
JP2016536132A (en) * 2013-09-09 2016-11-24 ラボ − オン − ア − ビード エービー Novel method and system for magnetic separation

Citations (5)

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Publication number Priority date Publication date Assignee Title
WO1996009379A1 (en) * 1994-09-20 1996-03-28 Whitehead Institute For Biomedical Research Dna purification and isolation using a solid phase
WO2001045522A1 (en) * 1999-12-20 2001-06-28 Ligochem, Inc. The removal of extraneous substances from biological fluids containing nucleic acids and the recovery of nucleic acids
US20030087286A1 (en) * 2000-09-06 2003-05-08 Hodge Timothy A. Isolation of eukaryotic genomic DNA using magnetically responsive solid functionalized particles
WO2004053115A1 (en) * 2002-12-09 2004-06-24 Tsinghua University Magnetism based rapid cell separation
US7022835B1 (en) * 1999-09-10 2006-04-04 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften. E.V. Method for binding nucleic acids to a solid phase

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996009379A1 (en) * 1994-09-20 1996-03-28 Whitehead Institute For Biomedical Research Dna purification and isolation using a solid phase
US7022835B1 (en) * 1999-09-10 2006-04-04 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften. E.V. Method for binding nucleic acids to a solid phase
WO2001045522A1 (en) * 1999-12-20 2001-06-28 Ligochem, Inc. The removal of extraneous substances from biological fluids containing nucleic acids and the recovery of nucleic acids
US20030087286A1 (en) * 2000-09-06 2003-05-08 Hodge Timothy A. Isolation of eukaryotic genomic DNA using magnetically responsive solid functionalized particles
WO2004053115A1 (en) * 2002-12-09 2004-06-24 Tsinghua University Magnetism based rapid cell separation

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016536132A (en) * 2013-09-09 2016-11-24 ラボ − オン − ア − ビード エービー Novel method and system for magnetic separation

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