Field of the invention:

This invention relates to a method for extraction of DNA using naked magnetic nanoparticles. More particularly, the invention relates to a method for DNA extraction from various biological systems using naked magnetic nanoparticles with high efficiency and purity.

Background of the invention:

DNA extraction has been a tool used extensively across the world in applied sciences. Most of the research breakthrough in biological research in the previous years directly or indirectly banked on DNA extraction. Applied research; be it Cloning or sequencing involves the use of DNA extraction methodologies. It was in the year of 1869 when Swiss scientist Friedrich Miescher first reported the extraction of DNA during a study conducted to elucidate the building blocks of life. Since then the science of extraction has advanced and has now reached its peak. A number of methods are available for DNA extraction in the literature viz. Phenol-chloroform, Benzyl alcohol method, Gradient based strategies, alkaline lysis method and so on. The newer approach in this regard is the solid phase extraction strategies. Solid-phase mentioned herein is primarily a physical moiety like glass beads, silica matrices etc. on which the DNA can bind and can be subsequently extracted. Recent advancements in this field involve the use of magnetisable solid phase support. This also serves an important purpose for the ease of extraction in the sense that the centrifugation step can be averted.

This strategy was first reported by Hawkins et. til. (Hawkins T L, O'Connor-Morin T, Roy A and Santillan C 1994, DNA purification and isolation using a solid-phase Nucleic Acids Res. 22: 4543^1). They proposed the use of carboxyl coated magnetic nanoparticles for extraction of DNA from bacterial cultures.

US Patent 5512439 discloses a method where oligonucleotides can be reversibly attached to microspheres impregnated with iron oxide and thereby target nucleic acids to be separated out from the surrounding media. US Patent 6027945 describes the use of magnetic silica particles with size ranging a few microns to extract and purify nucleic acids including DNA fragments, plasmid DNA, and R A.

WO 96/18731 by Deggerdal el. al. discloses a method where nucleic acid is extracted in the presence of a solid support by treating the samples with a detergent. The process results in the binding of DNA present in the sample to the support which can later be eluted out.

Use of naked magnetic nanoparticles was later reported by Saiyed et. o/.(Saiyed ZM, Ramchand CN, Extraction of genomic DNA using magnetic nanoparticles (Fe ₃ 0 _< i) as a solid-phase support, Am J Infect Dis 2007; 3: 225-9.) In this report, high salt and Polyethelyne glycol concentrations were employed to bind the DNA on naked magnetic nanoparticles which makes the method not useful for preparing ultra-pure DNA in good yield. The method is also disclosed in UK patent application GB 2455780.Thus the prior art contain methods that include various magnetic and non magnetic solid phases for the extraction of DNA. However they have certain disadvantages such as the use of instruments like centrifuge, heating blocks, use of organic solvents etc.

Therefore, there is a need in the art to come up with a robust method for DNA extraction where the naked magnetic nanoparticles specifically bind to DNA molecules from the biological milieu that can be efficiently eluted afterwards without compromising on yield and purity.

Accordingly, it is an objective of the present invention to provide an efficient and robust method for extraction of DNA from various biological systems using naked magnetic nanoparticles without compromising on the purity of the DNA extracted.

Another objective of the invention is to provide a method, where the damage done to the DNA during extraction is reduced to a bare minimum and further avoids the use of instruments like centrifuge etc. Summary of the invention:

To meet the above objectives, the present invention provides a method for the extraction of DNA from biological samples using naked magnetic nanoparticles with the help of a magnetic solid support which is preferably (but not limited to be) uncoated. The magnetic nanoparticle used according to the invention can be synthetic analogues of any suitable magnetic material or combination of materials, such as magnetite, ulvospinel, hematite, ilmenite, maghemite, jacobsite, trevorite, magnesioferrite, pyrrhotite, greigite, troilite, goethite,lepidocrocite, feroxyhyte, iron, nickel, cobalt, awaruite, wairauite, or any combination thereof. It can also be made up of transition metal such as iron, manganese, nickel, cobalt, zinc, etc. These magnetic particles can be of various shapes and sizes as the extraction is not dependent on any of these parameters.

The method disclosed in the present invention makes use of a proprietary mix of extraction solution which comprises magnetic nanOparticles in an amount of 1 to 3 mg/ml together with binding buffer consisting of monovalent salt in a concentration of 1 to 5 moles and PEG-8000 in an amount of 5 to 25% w/w of the total mixture. The use of the extraction solution separates the DNA from all proteins and hemoglobin thereby yield the resultant DNA with good yields and great purity.

In one aspect the current method can extract DNA from a number of biological samples including but not limited to Blood, Plant tissues, Animal tissues, Bacteria, Viruses, Fungi, Saliva, Urine, Stool, Semen, CSF, Paraffin fixed tissues, Tears, Amniotic fluid, Soil etc. The method makes use of specialized lysis buffer to lyse the cellular components for effectively exposing the DNA to magnetic support. The method also involves components which bind the DNA reversibly to magnetic support that could be effectively removed later for downstream processing like PCR amplification, Sequencing etc.

The method in another aspect contains special wash solutions and conditions whereby minimal contaminants are bound to the magnetic particles. Concurrently, the contaminants, if any, are effectively washed off from the magnetic nanoparticles without the loss of bound DNA on it. Detailed description of Figures:

Figure 1 : General protocol for the extraction of DNA from different biological systems using uncoated magnetic nanoparticles

Figure 2: DNA extracted from FFPE samples (Lane 1 , 2, 3 and 4 represent extraction from 3 different FFPE Samples)

Figure 3: DNA extracted from Fecal samples (Lane 1 and 2 represent extractions from fecal in duplicates)

Figure 4: PCR amplification of Fecal samples

Figure S: DNA extracted from Bacterial samples (Lane 1 , 2, 3, and 4 represent extraction from different bacteria)

Figure 6: PCR amplification of Bacterial samples

Figure 7: Sequencing chromatogram of the PCR product amplified from extracted DNA using Sanger sequencing technology

Figure 8: DNA extracted from Blood samples(Lane 1 , 2, 3 and 4 represent extraction from blood collected from 4 individuals)

Figure 9: PCR amplification of Blood samples

Figure 10: DNA extracted from Τΐ55με samples(Lane 1 , 2, 3, and 4 represent extractions from different tissues)

Figure 11 : PCR amplification of Tissue samples

Figure 12: DNA extracted from Saliva samples(Lane 1, 2, 3 and 4 represent extraction from saliva collected from 4 individuals)

Figure 13: PCR amplification of Saliva samples

Figure 14: Comparison of four different Tissue DNA samples extracted using Magnetic nanoparticle based method (to the left of the marker) and using conventional method (to the right of the marker)

Figure 15: Comparison of four different Plant DNA samples extracted using Magnetic nanoparticle based method (to the left of the marker) and using conventional method (to the right of the marker)

Figure 16: Comparison of four different Blood DNA samples extracted using Magnetic nanoparticle based method (to the left of the marker) and using conventional method (to the right of the marker) Figure 17: Comparison of four different Bacterial DNA samples extracted using Magnetic nanoparticle based method (to the left of the marker) and using conventional method (to the right of the marker)

Figure 18 depicts comparison of DNA extracted from blood samples using (a) Method mentioned in GB 2455780 patent and DNA extracted using the (b) present method (All proteins and haemoglobin removed

Figure 19 depicts comparison of DNA Purity and yield of DNA extracted from Blood using (a) Method mentioned in GB 2455780 patent and (b) DNA extracted using the present method (All proteins and haemoglobin removed), which indicates that the present method gives recovery almost double than the method disclosed in GB'780. The 260/280 and 260/230 ratios of around 1.8 shows pure DNA and not contaminated by RNA and/or proteins.

Figure 20 depicts DNA extracted using (a) Method mentioned in GB 780 (2009) patent and DNA extracted using the (b) present method (higher recovery)

Detailed description of the invention:

The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

Accordingly, the instant invention provides an efficient method for extracting DNA from various biological samples like Formalin fixed and paraffin embedded (FFPE), Fecal, Bacteria, Blood, Plant tissue, animal Tissues, Viruses, Fungi, Saliva, Urine, Stool, Semen, CSF, Paraffin fixed tissues, Tears, Amniotic fluid, Soil etc. using naked magnetic nanoparticles. The general protocol for the extraction is explained in Figurel . In a preferred embodiment, the method for extracting pure DNA from various biological samples comprises of :

1. Initiating lysis by incubating the sample in suitable lysis buffer;

2. Adding a proprietary mix of extraction solution consisting of magnetic nanoparticles and binding buffer to the lysed sample so as to precipitate DNA and thereby allowing the magnetic particles to capture on same; 3. Subjecting the precipitated DNA loaded-magnetic nanoparticles to the magnetic field so as to settle down the DNA adsorbed nanoparticles from the supernatant containing the contaminants;

4. Washing the settled DNA using a wash buffer followed by elution using TE buffer or nuclease free water.

The method disclosed in the present invention makes use of a proprietary mix of extraction solution which comprises magnetic nanoparticles in an amount of 1 to 3 mg/ml together with binding buffer consisting of monovalent salt in a concentration of 1 to 5 moles and polymer such as PEG-8000 in an amount of 5 to 25% w/w of the total mixture. The use of the extraction solution separates the DNA from all proteins and hemoglobin thereby yield the resultant DNA with good yields and great purity.

Depending on the sample to be lysed, the lysis buffer contains combinations of various detergents such as alkali metal alkylsulphate salt, Urea and Tween 20 or combinations thereof. One preferred alkali metal alkylsulphate salt is sodium dodecyl sulphate.

One preferred lysis buffer for a biological sample of Blood comprises of Sodium dodecyl sulphate and Tween 20. The incubation for lysis typically takes 3-5 minutes. In some of the embodiments of the invention, the lysed tissue optionally incubated with Proteinase or RNase enzymes or both.

Thus the present invention encompasses the proprietary mix of extraction solution which comprising of magnetic nanoparticles and binding buffer consisting of a monovalent salt and a polymer. The proprietary mix of extraction solution makes the process more efficient and contamination free. The magnetic nanoparticles are preferably magnetite (Fe304) while the binding buffer contains polyethylene glycol and a monovalent salt. Thus the magnetic nanoparticles of size within 10-lOOnm are more readily available for binding the DNA and the action is more specific in the sense that it prevents non-specific adsorption of other cellular biomolecules.

In an embodiment, the naked magnetic nanoparticles are selected from the group consisting of oxides of synthetic analogues of any suitable magnetic material or combination of materials, such as magnetite, ulvospinel, hematite, ilmenite, maghemite, jacobsite, trevorite, magnesioferrite, pyrrhotite, greigite, troilite, goethite, lepidocrocite, feroxyhyte, iron, nickel, cobalt, awaruite and wairauite.

The naked magnetic nanoparticles used, according to the invention are preferably divalent or trivalent iron oxide. One preferred iron oxide nanoparticles are magnetite (Fe30,() magnetic nanoparticles, preferably of size ranging from 10-lOOnm. The magnetic nanoparticles are preferably uncoated.

According to another embodiment, the binding buffer contains polyethylene glycol and a monovalent salt. The monovalent salt used in the binding buffer for the extraction of DNA is selected from the group consisting of Sodium chloride, lithium chloride, Potassium chloride or a mixture thereof. The monovalent salt is used in 1 to 5 molar concentration of the mixture.

The polyethylene glycol or polyoxyethylene-8000 (POE) is used in the concentration between 5 to 25 % of the mixture.

In a preferred embodiment, the proprietary mix of extraction solution comprises:

a) Magnetic nanoparticles 1 to 3 mg/ml;

b) PEG-8000 in an amount of 20 to 25%; ^'

c) NaCl- 1 to 5molar,

wherein, the pH of the extraction solution is maintained in the range of 6.0 to 8.0 ± 0.2. In a more specific embodiment, the proprietary mix of extraction solution comprises: a) Magnetic nanoparticles 1 to 3 mg/ml;

b) PEG-8000 in an amount of 20%;

c) NaCl- 4 molar concentration,

wherein, the pH of the extraction solution is maintained at 8.0 ± 0.2.

The recovery of the DNA is higher in the case of the method disclosed in the invention over the method disclosed in the patent GB 2455780. A comparative example of the method of GB'780 vis-a-vis the present invention that employs the specific proprietary mix of extraction solution for the recovery of pure DNA from blood sample without protein and haemoglobin contamination is described in figures 18. The spectra and 260/280 and 260/230 ratios are ideal in the case of present method. The comparison between the yields of the present and the method disclosed in GB'780 is shown in figure 20. Effective removal of protein is shown in figure 18 and spectra with 260/280 and 260/230 ratios are shown in figure 19. The specific proprietary mixture removes proteins, haemoglobin, RNA and small molecules, especially in blood samples the haemoglobin contamination is completely removed as shown in figure 20.

From the above, it is evident that the present method gives recovery almost double then the previous method disclosed in GB'780. The 260/280 and 260/230 ratios of around 1.8 show pure DNA not contaminated by RNA and/or proteins.

The extraction of lysed DNA using proprietary mix of extraction solution and the components with specific concentration according to the invention reduces the damage done to the DNA during extraction and thus results in good yield and purity. The present invention further eliminates the use of centrifugation instruments etc.

An important feature of the invention includes, the provision of proprietary mix of magnetic nanoparticles and the binding buffer together in a proprietary mix which makes the process more efficient and contamination free. The magnetic nanoparticles( of size within 10- 1 OOnm) are more readily available for binding the DNA and the action is more specific in the sense that it prevents non-specific adsorption of other cellular biomolecules.

The specialized two part wash buffer used in the instant invention removes contaminants and keeps the DNA tightly bound to the nanoparticles concurrently. The wash buffers used in the invention are based on ethanol and other organic solvents such as acetone.

After washing, the adsorbed DNA is incubated at mild temperatures for 5 to 10 minutes prior to elution from the nanoparticles. This could be done by subjecting the pellet to hydrophilic conditions. The elution buffer according to the invention may be, for example, TE buffer (Tris-EDTA buffer) or nuclease free water.

Thus the method for extracting DNA from a biological sample, according to the invention is initiated with the addition of a suitable lysis buffer. The lysis buffer depends on the type of sample used. For example, for Blood sample, the lysis buffer contains a combination of detergents preferably Sodium dodecyl sulphate and Tween 20. Post to the addition of the lysis buffer, there is an incubation for some time, preferably 3 to 5 minutes. The binding buffer containing the magnetic nanoparticles is added to the system thereafter. The binding buffer forces the system towards a hydrophobic milieu thereby causing the DNA to precipitate. The magnetic nanoparticles present in the system therefore serves as a site for this adsorption. A mentionable aspect here is that, unlike most protocols the magnetic nanoparticles and the binding buffer are clubbed together in a proprietary mix which makes the process more efficient and contamination free. As mentioned above the magnetic nanoparticles are preferably magnetite (Fe ₃ 0 ₄ ) while the binding buffer contains polyethylene glycol and a monovalent salt. Therefore the magnetic nanoparticles (Size within 10-lOOnm) are more readily available for binding the DNA and the action is more specific in the sense that it prevents non-specific adsorption of other cellular biomolecules. This step is followed by subjecting the system to an external magnetic field where the pellet of DNA-adsorbed nanoparticles settle down under this effect and the supernatant containing the contaminants can be subsequently discarded. The pellet is formed at the bottom or the side of the tube depending on the direction of the field. The pellet still needs to be washed thoroughly to obtain an ultra-pure DNA. In most methods when the DNA adsorbed -Magnetic nanoparticles pellet is washed, the adsorbed DNA gets detached easily. This can cause a 30 to 70 % decrease in the yield. On the contrary in the instant method a specialized wash buffer is used to remove contaminants and keeps DNA tightly bound to the nanoparticles concurrently. For instance, the wash buffer used in the invention is a two part wash buffer in which one contains 80% ethanol and other contains ice-cold acetone. The DNA can be subsequently eluted from the nanoparticles in an elution buffer by subjecting the pellet with hydrophilic conditions. The elution buffer is TE buffer or nuclease free water.

In another embodiment, the invention provides a method for extraction of DNA from Formalin fixed paraffin embedded tissues (FFPE) using magnetic nanoparticles that gives DNA with high integrity and purity which comprises:

a) Scrapping FFPE tissue from paraffin block followed by washing with an aromatic hydrocarbon mixture preferably Xylene and subsequently rinsed with ethanol; b) lysing the tissue using a detergent based lysis buffer followed by incubating with

Proteinase enzymes; c) treating the lysed sample with proprietary mix of extraction solution according to claim 1, to precipitate DNA and thereby allowing the magnetic particles to capture on same;

d) subjecting the precipitated DNA loaded-magnetic nanoparticles to the magnetic field to settle down the DNA adsorbed nanoparticles in the form of a immobilized pellet from the supernatant containing the contaminants; and e) Washing the pellet with ethanol followed by eluting the DNA by TE buffer with heating at the temperature ranging from 90 °C.

In a further embodiment, the invention provides a method for extraction of DNA from Faecal and Soil samples using magnetic nanoparticles that gives DNA with high integrity and purity which method comprises;

a) Treating the sample with lysis buffer containing alkali metal alkylsulphate salt, more preferably sodium dodecyl sulphate followed by incubating with Proteinase and RNase enzymes;

b) Treating the lysed samples with proprietary mix of extraction solution according to claim 1, to precipitate DNA and thereby allowing the magnetic particles to capture on same;

c) Subjecting the precipitated DNA loaded-magnetic nanoparticles to the magnetic field to settle down the DNA adsorbed nanoparticles in the form of a immobilized pellet from the supernatant containing the contaminants;

d) Discarding the supernatant and washing the pellet with wash buffer containing salts and ethanol followed by eluting DNA by adding TE buffer with heating at the temperature ranging from 50°C to 60°C; and

e) Optionally subjecting the eluted DNA for rebinding with the proprietary mix of extraction solution and repeating the steps (c) to (d) to obtain ultra- pure DNA.

The lysis buffer used in accordance with the invention comprises alkali metal alkylsulphate salt, more preferably sodium dodecyl sulphate.

Thus in a further embodiment, the invention provides a kit for extraction of ultra-pure DNA with high yield from a biological sample which comprises;

a) Proprietary mix of extraction solution consisting of magnetic nanoparticles, monovalent salt and polyethylene glycol in a specific combination; b) Lysis buffer containing combination of detergents;

c) Two part wash buffer consisting of ethanol and acetone;

d) Elution buffer consisting of TE buffer or nuclease free water.

The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention.

Example 1

Extraction of DNA from FFPE

Formalin fixation and paraffin embedding (FFPE) is a standard method for long-term preservation of most archived pathological specimens. Such samples provide an invaluable source for subsequent molecular studies of clinical phenotypes, especially genetic studies in which DNA is not available from fresh or frozen tissue(s) because the subjects are no longer alive. FFPE tissue is an excellent source of DNA, but its extraction remains a challenge. Formaldehyde, the effective component of formalin, leads to the generation of cross-linking between nucleic acids and proteins, and causes nucleic acids to fragment because of fixation process conditions. To extract DNA from paraffin- embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment; which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion.Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase during the incubation period of 30minutes. Once lysis is over, the tissue can be subjected to the protocol mentioned in Figure 1 which leads to a shear free high recovery of DNA. The binding buffer which contains the nanoparticles, efficiently bind the entire DNA and subsequently releases it later in the elution buffer. Elution of DNA is done at 90 °C. The results for extraction from different FFPE samples are depicted in Figure 2.

Example 2

Extraction of DNA from Fecal samples

Fecal sampling is regarded as an effective, non-invasive and easy method for various genetic and ecological research.The samples are to be lysed in a suitable lysis buffer containing combination of detergents preferably Sodium dodecyl sulphate and Tween 20. Protein and RNA contaminants are removed by incubating the sample with Proteinase K and RNase A respectively. Lysis is followed by incubation with iron magnetic nanoparticles and washing using suitable wash buffers, after which the bound DNA is eluted by adding TE buffer/Nuclease free water with heating at the temperature ranging from 50 °C to 60°C. The result of extraction is shown in Figure 3. The PCR amplification of extracted DNA samples have proved that the extracted DNA is amenable to downstream appIications.The amplification results are shown in Figure 4.

Example 3

Extraction of DNA from Bacteria

Bacterial cell wall makes the process of DNA extraction difficult; moreover there is always a chance of high RNA contamination. On the contrary the current method minimizes the RNA contamination and the lysis buffer, containing a suitable surfactant causes complete lysis of cells apart from keeping the DNA intact. The method is efficient for both gram positive and gram negative bacteria. The cells are harvested and washed once with PBS. After the pellet is formed, the rest protocol is same as shown in Figure 1. The results for extraction of DNA from bacterial system are shown in Figure 5 (Extracted DNA) .To test the amenability of extracted DNA for general post processing applications of DNA, the Bacterial DNA sample was subjected to amplification (16S-rRNA) and subsequently for sequencing. The results show that the method of extraction is amenable for post processing applications of DNA. The amplification results are shown in Figure 6 and the sequencing chromatogram in Figure 7.

Example 4

Extraction of DNA from Blood

Blood is used as an effective source for the extraction of DNA. Tn spite of being a consistent system, a number of challenges remain for efficient extraction of DNA. Various complexities like large amount of protein content, presence of RBCs etc poses major issues and demands a potential protocol for isolation. The highlight of the study remains mainly in the simplicity of the method, time, cost, number of reagents used, instrumentation etc. The environment created by the buffering systems ensures the molecular integrity and intactness of the extracted DNA along with purity and concentration. The buffers constitute a suitable chelating agent, surfactants and detergents which help in membrane solublization, digestion etc. In the current invention, a starting sample volume of 200μ1 of blood is used. For the removal of RBCs, a buffer solution containing EDTA is used. The WBC lysis is attained by the addition of detergents like SDS, Tween-20 and Urea followed by incubation for efficient digestion. A binding solution with specific mixture of PEG-8000 and 4M NaCl is used for the suspension of magnetic nanoparticles (1 to 3 mg ml) which creates a hydrophobic environment for DNA precipitation. Washing is carried out using 80% Ethanol and Acetone followed by sample elution by adding TE buffer/nuclease free water with heating at the temperature ranging from 50 °C to 60°C;

The method yielded a DNA concentration of about 4μg with a 200/280 ratio ranging between 1.7 and 1.9 which is shown in Figure 8. Figure 9 shows amplification using Human primers.

Example S

Extraction of DNA from Tissue

Tissue DNA isolation protocol helps in the isolation of DNA from about 50-100mg of starting material. Various methods of tissue homogenization can be adopted like crushing the tissue in liquid nitrogen, mincing or even directly dropping the sample in the lysis buffer. The method of homogenization determines the time taken for extraction. Lysis is facilitated by a suitable buffer containing Tris-HCl, EDTA, CaC¾, SDS, and Urea. Enzymatic digestions using RNase A and Proteinase K is also adopted for better yields and purity. A magnetic nanoparticle binding solution comprising of PEG-8000 and NaCl is added to the digested sample facilitating DNA binding. Washes using 80% Ethanol is carried out to remove any salts and other contaminants. DNA is finally eluted by adding TE buffer/nuclease free water with heating at the temperature ranging from 50 °C to 60°C Depending on the tissue used, the yield of DNA varies from sample to sample which ranges between 20-40μg of DNA with a purity of 1.7-1.9 which is shown in Figure 10. Amplification using Human primers is shown in Figure 11. Example 6

Extraction of DNA from Saliva

DNA isolation from saliva has been regarded as a non-invasive yet efficient way of DNA extraction. A large number of studies selectively prefer saliva as the source for DNA isolation. The protocol followed here is relatively simple and quick and yields considerable concentration of DNA with good ρωΊΐν.500 1 Saliva sample is incubated in a lysis buffer containing Ca ¾, SDS and Tris-HCl. The cellular contents are again incubated with magnetic nanoparticles in a buffer medium containing PEG-8000 and NaCl. The DNA bound magnetic nanoparticles are washed using 80% Ethanol and Acetone, by adding TE buffer/nuclease free water with heating at the temperature ranging from 50 °C to 60°C

The eluted DNA was found to be around 2 g in concentration with a purity ratio between 17.-1.9 which is shown in Figure 12. Amplification using Human primers is shown in Figure 13.

Thus the current method serves as an excellent approach for quick and efficient extraction of DNA from various biological systems like Bacteria, Fecal, FFPE, Blood, Plants, Tissues, Viruses, Fungi, Saliva, Urine, Stool, Semen, CSF, Tears, Amniotic fluid, Soil etc. The method is based mainly on effectively lysing the cells followed by addition of specific magnetic nanoparticle containing binding buffer. This provides conditions where DNA specifically binds to the naked nanoparticles effectively. The specialized wash buffers used thereafter cleans any remaining contamination without downgrading the yield of DNA. The DNA is finally eluted with high quantity and high purity in the elution buffer.

Efforts to extract DNA from Soil, Water and also purifying the desired range of DNA fragment have also found success. The protocol follows the same principle of extraction where modifications are made in certain steps depending on the sample to be processed. In case of extracting DNA from both soil and water, the sample is subjected to heat shock followed by enzyme treatments using Proteinase K and RNase A. The rest protocol, i.e, binding and subsequent washes remain the same. In the case of Size exclusion, the DNA sample to be purified is mixed with appropriate binding buffer containing nanoparticles. The concentration of magnetic nanoparticles is characteristic of the size of the DNA fragment to be purified.

The advantages of the method disclosed in the instant invention are:

• High purity extraction procedure with greater recovery and ease

• Process is completed in 30-40 minutes post lysis step

• Method do not involve use of a centrifugation step

• Extracted DNA amenable for various downstream applications

• Use of specialized magnetic nanoparticle containing binding buffer, specialized lysis buffer and potent wash solution ensuring that the DNA extraction is quantitatively and qualitatively superior.