1. A novel process for extraction of DNA, said process comprises the steps of: (a) pre-treating a sample to remove non-polar substances from the sample to obtain pre-treated sample, (b) mixing said pre-treated sample with an extraction buffer, (c) precipitating said mixture of step (b) to obtain pellet of DNA (PI), (d) washing PI with a washing solution and centrifuging to obtain purified and air dried pellet of DNA (P2), (e) storing said P2 in a buffer solution/elution buffer at specific temperature, characterized in that said pre-treating of step (a) is carried out by a non-polar solvent; said extraction buffer in step (b) is Tris HC1; said precipitation step comprises pre-defined steps of reactions with specific reagents under specific reaction conditions and pre-defined steps of centrifugation under specific conditions to obtain pellet of DNA (PI), said process enables extraction of small quantity of DNA from even traces of a highly processed sample.

2. The process for extraction of DNA as claimed in claim 1, wherein said solvent for pre-treatment of sample is selected from acetone or hexane or toluene.

3. The process for extraction of DNA as claimed in claim 1, wherein said solvent is hexane.

4. The process for extraction of DNA as claimed in claim 1, wherein said precipitation step comprises of: (a) mixing said pre-treated sample (P) with the extraction buffer at pH 8, (b) vortexing of said mixture of step (a) vigorously for 90 minutes to obtain a uniform solution (SI), (c) centrifuging said solution of step (b) (SI) at 13000 rpm for 25 minutes at room temperature (RT) (CI), (d) transferring aqueous phase of the CI (Al) in 2 ml centrifugation tube and again centrifuging at 13000 rpm for 25 minutes at RT (C2), (e) transferring aqueous layer (1 ml) of the C2 (A2) in 1.5 ml centrifugation tube and adding chloroform (500 ul) and vortexing vigorously for 2-3 minutes till the solution turns milky to obtain solution S2, (f) centrifuging the solution S2 at 13000 rpm for 15 minutes at RT (C3), (g) transferring upper aqueous phase (700 ul) of C3 (A3) to 1.5 ml centrifugation tube and adding 0.8 volumes of isopropanol (560 ul) and 4 ul glycogen of 3 mg/ml and gentle mixing by inverting the tube for 4-5 times to obtain solution S3, (h) incubating the S3 at room temperature for 30 minutes to obtain incubated solution S4, (i) centrifuging the tube containing S4 at 14000 rpm for 20 minutes at RT to obtain pellet (PI), (j) pipetting out isopropanol without disturbing the pellet (PI) and air drying of the pellet (PI).

5. The process for extraction of DNA as claimed in claim 1, wherein said air dried pellet (PI) is washed with washing buffer and centrifuged at 14000 rpm for 20 minutes at RT to obtain purified pellet (P2), while the washing buffer is removed and the pellet (P2) is air dried to store in the elution buffer.

6. The process for extraction of DNA as claimed in claim 1, wherein said washing buffer is 70% ethanol.

7. The process for extraction of DNA as claimed in claim 1, wherein said elution buffer is Tris EDTA.

8. The process for extraction of DNA as claimed in claim 1, wherein said air dried purified DNA pellet (P2) is dissolved in TE (Tris-EDTA) buffer (25 ul) and stored at -20°C.

9. The process for extraction of DNA as claimed in any of the preceding claims wherein the process is further comprises of purification step if purified pellet is not obtained or is coloured due to the presence of impurities, wherein said purification step comprises of repetition of precipitation steps from 3(e) to 3(j) steps of precipitation followed by washing and storing of air dried and purified DNA in buffer solution.

10. The process for extraction of DNA as claimed in claim 1 wherein said process is highly sensitive, economical and simple.

11. A process of extraction of DNA from lecithin sample, said process comprises the steps of: (a) pre-treating the lecithin sample with hexane to remove non-polar substances to obtain pre-treated sample (Pr), (b) desolventization of pre-treated sample (Pr) by removing the hexane, (c) mixing said pre-treated and desolventized sample with Tris HC1 lOmM, (d) vortexing of said mixture of step (c) vigorously for 90 minutes to obtain a uniform solution (Sri), (e) centrifuging said solution of step (d) (Sri) at 13000 rpm for 25 minutes at 25°C (Crl), (f) transferring aqueous phase of Crl (Arl) in 2 ml centrifugation tube and again centrifuging at 13000 rpm for 25 minutes at 25°C (Cr2), (g) transferring aqueous layer (1 ml) of Cr2 (Ar2) in 1.5 ml centrifugation tube and adding chloroform (500 ul) and vortexing vigorously for 2-3 minutes till the solution turns milky to obtain solution Sr2, (h) centrifuging the solution Sr2 at 13000 rpm for 15 minutes at 25°C (Cr3), (i) transferring upper aqueous phase (700 ul) of Cr3 (Ar3) to 1.5 ml centrifugation tube and adding 0.8 volumes of isopropanol (560 ul) and 4 ul glycogen (3 mg/ml) and gentle mixing by inverting the tube for 4-5 times to obtain solution Sr3, (j) incubating the Sr3 at room temperature for 30 minutes to obtain incubated solution Sr4, (k) centrifuging the tube containing Sr4 at 14000 rpm for 20 minutes at 25°C to obtain pellet (Prl), (1) pipetting out isopropanol without disturbing the pellet (Prl) and air drying of pellet (Prl), (m)washing Prl with washing buffer and centrifuged at 14000 rpm for 20 minutes at 25°C to obtain purified pellet (Pr2), while the washing buffer is removed and the pellet (Pr2) is air dried to store in Tris EDTA at -20°C.

12. The process for extraction of DNA as claimed in claim 11, wherein said lecithin is rice bran lecithin.

13. A kit for extraction of DNA, said kit comprising: i. Pre-treat, said pre-treat comprising a solvent for pre-treatment of sample, said solvent being selected from a non-polar solvent, ii. Solution A comprising an extraction buffer, said extraction buffer comprises of Tris HC1 of pH 8 of concentration range from lOmM to 20 mM, iii. Solution B comprising a chloroform solution, iv. Solution C comprising Solution CI and C2, wherein Solution CI comprises of isopropanol and solution C2 comprises of glycogen in the concentration of 3 mg/ml, v. Washing solution comprising 70% ethanol. vi. Buffer solution or Elution buffer comprising Tris-EDTA. wherein said kit is economic and free from any salt of reagents causing interference in the amplification of isolated DNA.

14. The kit for DNA extraction as claimed in claim 13, wherein said Pre-treat solvent is selected from hexane or acetone or toluene.

15. The kit for DNA extraction as claimed in claim 13, wherein said Pre-treat solvent is hexane.

16. The kit for DNA extraction as claimed in claim 13 wherein said Tris HCl is of lOmM concentration.

FIELD OF THE INVENTION
The present invention relates to the field of molecular biology. The present invention relates to a novel process for extraction of DNA. In particular, the present invention relates to extraction of small quantity of DNA from a highly processed sample. The process of the present invention is highly sensitive, economical and simple process of DNA extraction from small amount of sample. The present invention also relates to a kit for extracting DNA from highly processed biological samples and subsequent amplification of the extracted DNA.
BACKGROUND OF THE INVENTION
Establishing authenticity of species origin is an important aspect of product quality control. It is also relevant from protection of consumer health and economic perspective. Most of the analysis methods target proteins or DNA molecules of the species to trace the origin. The DNA/proteins are extracted from the test sample. However, the commercial end products are subjected to various physiochemical and thermal attritions such as thermal mechanical disruption, lysis, canning, heating, etc. during the production process. Due to this, the proteins in the product get denatured and hence DNA is the moiety of choice as suitable molecular marker for species authentication. The DNA isolated from samples is purified and amplified for the detection and identification of species or unwanted organisms.
The quality and quantity of DNA plays a crucial role in most of the amplification based analysis. Therefore, the extraction and purification of the DNA is a critical step for species identification in samples. Low amounts of nucleic acids (DNA, RNA) are present in highly processed samples which are subjected to enzymatic, mechanical and chemical treatments during industrial production. The quality of DNA is influenced by various factors such as the presence of PCR inhibitors, degree of DNA damage, average length of the fragment of nucleic acid, pH. At acidic pH, depurination takes place which subsequently leads to the nick formation in DNA.

The extraction efficiency depends on the length of the fragmented DNA, different physicochemical changes which occur during the processing, oxidation, binding efficiency of fragmented DNA to the resins or insoluble matrix components and enzymatic hydrolysis etc.
Different methods available for the DNA extraction are based on precipitation. There are kit-based methods used for DNA extraction which include DNeasy method, Wizard method (Promega, USA) and Nucleon Phytopure kit method (Scotlab Bioscience, USA). In DNeasy kit method, DNA is extracted using API (lysis buffer), AP2 (protein precipitation buffer) and AP3 (binding buffer) along with proteinase K (20 mg/ml). In Wizard method (Promega, USA) TNE buffer is the extraction buffer having 1% SDS. Apart from extraction buffer, 100 ul 5M guanidium hydrochloride is added for DNA isolation. In Nucleon Phytopure kit method (Scotlab Bioscience, USA) Reagent 1 and Reagent 2 are used for extraction. Nucleon Phytopure DNA extraction silica suspension was added after chloroform treatment. These kit based methods result in low recovery of DNA (<20%) and are expensive. Each kit contains different extraction buffers with different composition. These methods are resin-column based leading to increase in the cost.
The other methods include ROSEX, Alkali X and Chelex 100. The ROSEX method includes ROSE buffer and 5% Chelex 100. In AlkaliX method neutralisation buffer (0.25% Nonident P-40 and 500 mM Tris-Cl, pH 8) along with 5% Chelex 100 is used. In Chelex 100 method IX TNECH buffer, 5M guanidium hydrochloride and proteinase K is used. Among these methods, ROSEX leads to partial DNA denaturation whereas Chelex 100 and Alkali X lead to complete denaturation of DNA. High DNA yield is obtained by these methods, but high concentrations of DNA lead to inhibition of the PCR system due to presence of various PCR inhibitors which are eluted along with DNA.

The most common method used for the extraction of DNA from lecithin samples includes Cetyl trimethyl ammonium bromide (CTAB) method. The extraction buffer contains 50 mM CTAB, 1.4 M NaCl, 0.8-1 M Tris-Cl, and 20 mM EDTA. Lecithin is thoroughly mixed with extraction buffer and incubated at 65°C. Further extraction and purification steps are carried out to isolate DNA.
Wurz et al., 1998 used large amount of guanidium isothiocyanate in the extraction buffer for DNA isolation. In this method, 2 gm lecithin is taken as the starting material and glycogen is used to precipitate very small amount of DNA.
These methods utilize different salts and detergents for cell lysis. The samples contain various contaminants that inhibit the process of amplification. The most common contaminants are polysaccharides, polyphenols, and lipids. Due to high lipid content present in rice bran lecithin, it becomes difficult to isolate DNA. Apart from these, other contaminants are present which include chemicals like phenol, SDS, CTAB, EDTA etc. which cause inhibition during the PCR process. The inhibitors inhibit the Taq Polymerase during the PCR process. The use of different chemicals and salts also reduces the PCR performance.
CN101575597 discloses a kit which is suitable for PCR amplification and used for quickly extracting plant genomes, comprising a lysate, a neutralizer and a precipitation liquid. The kit improves and optimizes the traditional alkaline lysis method used for preparing genome and enlarges the applicable range of the alkaline lysis method used for preparing genome. The kit is widely applied to the extraction on the genome of various plants and can quickly extract the genome used for PCR template directly from plant seeds. The kit has obvious detection effect on the target gene of 600bp, has excellent amplification effects on endogenous gene and exogenous gene and can discover the samples with the components to be measured

more than 1%. The extraction time for the whole genome is controlled within 21min, thus greatly shortening the extraction time of the genome.
DE102014103107A1 discloses a composition, method and kit for the simplified and safe extraction of nucleic acids from biological samples, such as raw or processed foods and subsequent analysis by polymerase chain reaction to the presence of animal matter, genetically modified organisms, allergens and pathogens. The method comprises adding an extraction composition together with hot water to extract and stabilize nucleic acids and to remove substances that interfere with DNA polymerase activity. The composition for use in extracting and purifying nucleic acids from food samples is a solid mixture comprising: 10 to 40% by weight of particles consisting of water-insoluble hydrogenated magnesium silicate and having an average particle size of 0.8 to 2.5 urn exhibit; and 20 to 70 weight percent crystalline phosphate buffered saline which is readily water soluble, thereby producing a solution having a pH of 5.5 to 7.0 at 70 to 95 degrees Celsius.
CN109055362A discloses a kind of kit and method for extracting long root mushroom genomic DNA. The composition includes: (1) equilibrium liquid: l%Na2Si03-9H20, l%NaOH ; (2) lysate: 5.0M guanidine hydrochloride, 25% isopropanol and 1%PVP K30 ; (3) cleaning solution: 20mMNaCl, 20 mM Tris-HCl, 85% ethyl alcohol, pH7.5 ; (4) eluent: lOmM Tris-HCl, pH8.5 ; (5) DNA adsorption column. The present invention extracts the noxious materials such as the reagent reactive phenol of long root mushroom genomic DNA, chloroform. It is safer, simple and convenient extraction with high efficiently and less extraction time of 15min.
CN103361339A discloses a filamentous fungus DNA extraction test kit comprising of solution A, solution B, solution C, solution D and DNA adsorption column, wherein, Solution A: 10-50mM Tris-HCl, 10-50mM EDTA-2Na, 1%-10%W/V SDS,

2-4M NaCl, the beta-mercaptoethanol of 0.5%-2%v/v, pH8.0; The Guanidinium hydrochloride of solution B: 2.5-6M or guanidinium isothiocyanate, 20%-50%v/v Virahol or dehydrated alcohol, 5mM-20mM EDTA-2Na, pH 4.5-6.5; The dehydrated alcohol of solution C: 70%-80%v/v, pH4.5-6.5; Solution D: the RNA enzyme aqueous solution of 5-50 [i g/mL, pH is transferred to <8.5 alkalescence with 1M NaOH solution.
CNl07267498A discloses DNA kit and method. DNA is extracted in a kind of universal material from trace plant. The kit comprises Extraction buffer (API) containing 2%CTAB, lOOmM Tris-HCl (pH8.0), 20mM EDTA-Na2, 0.5-1.5M NaCl, 2-5% PVP, 1-3% ascorbic acid; Silicon matrix post combination liquid (AP2, AP3) wherein AP2 comprises 2.5-4.5M guanidine hydrochlorides, 0.5-1.5M sodium acetates and AP3 comprises 40-60% absolute ethyl alcohols, 0.5-1.5M sodium acetate; impurity removes liquid (APW) of 70-80% absolute ethyl alcohols; DNA eluents (APE) of lOmM Tris-Cl, 5mM EDTA-Na2(pH8.5).
CN105087550A discloses method for rapid and high-flux extraction of plant genome DNA and application of plant genome DNA. The quick, high-throughput method comprises of:
(1) get plant tissue to be extracted and add the mixing of SDS extracting solution,
and fully grind; Described SDS extract recipe is: 80-120mMTrisHCl, 40-
60mMEDTA, 80-120mMNaCl, 0.5-1.5%w/vSDS, pH8.0;
(2) sample after grinding is heated 5 - lOmin in 60-70 DEG C, and high speed centrifugation process under room temperature; Pipette samples supernatant liquor subsequently, and add the mixing of isopyknic Virahol, left at room temperature process;
(3) by the high speed centrifugation process again of the sample after standing process, and remove supernatant liquor, add the ethanol that mass concentration is not less than 70% subsequently, fully mix;

(4) by the high speed centrifugation process again of mixed sample, after complete
abandoning supernatant, by described sample drying process;
(5) in dried sample, add distilled water, leave standstill at 35-40 DEG C of
temperature, and routine preservation, obtain required plant genome DNA and
carry out follow-up PCR reaction as template.
CN110358762A discloses a kit for extracting plant leaf blade genomic DNA, the kit comprising of Pretreat, Lysis A, Lysis B, bead suspension, Binding Buffer, Washing Buffer and Elution Buffer. The component of the Pretreat are 0.1-0.3M Tris-HCl, 0.04-0.06M EDTA, 1-3% P-sulfydryl second Alcohol, 1-2% PVP; the component of the Lysis A are 0.8-1.2M KAC, 0.04-0.06M EDTA, 4-6M GHCL, 5~20mM Sodium citrate, l~2%Tween 20; the component of the Lysis B are 20~30mg/ml Proteinase K; the component of the bead suspension are magnetic bead concentration (lOOmg/ml); the component of the Binding Buffer are 15~25%PEG6000,1.0~2.0M NaCl, pH=4.0-5.0; the component of the Washing Buffer are 70-80% ethyl alcohol; the component of the Elution Buffer are sterile ultrapure water, pH=7.0.
The critical step in these approaches is the extraction of high-quality DNA in sufficiently large quantities from small quantities of highly processed samples. Due to presence of small DNA quantities in processed samples because of different factors, it becomes difficult to isolate the integrated full length DNA. Lack of proper purification leads to interference of contaminants with the amplification process. For example, chemicals and salts used during DNA extraction remains attached to the DNA which leads to the PCR inhibition. Therefore, new methods which are feasible at the industrial level must be used which lead to the DNA yield with minimum amount of PCR inhibitors (proteins, salts, lipids etc.).

In order to obviate the drawbacks in the existing state of the art, the present invention provides a simple and economical process of extraction of DNA from samples. The present invention does not employ the detergents like SDS or CTAB, salts which bind to DNA. The invention is useful for DNA extraction from the samples where DNA is present in the scattered form (not entrapped inside the cell). This condition occurs in highly processed samples where only traces of DNA are left in the sample due to breaking of cells containing DNA. The present invention also provides a simple and economic kit for DNA extraction from highly processed sample.
OBJECTS OF THE INVENTION
Accordingly, the main object of the present invention is to provide a novel process for extraction of DNA from a sample.
Another object of the present invention is to provide a highly sensitive process for extraction of small amount of DNA from traces of a highly processed sample.
Yet another object of the present invention is to provide a highly sensitive process to extract a highly purified DNA from traces of a highly processed sample.
Yet another object of the present invention is to provide an economical and simple process for extraction of purified DNA from small amount of a highly processed sample.
Yet another objective of the present invention is to quantitatively and qualitatively assess the isolated DNA from highly processed sample.
Yet another object of the present invention is to provide a kit for extracting DNA from highly processed samples and subsequent amplification of the extracted DNA.

SUMMARY OF THE INVENTION
It will nevertheless be understood that no limitation of the scope of the invention is thereby intended by way of embodiments and examples. Such alterations and further modifications in the present invention, and such further applications of the principles of the invention as would normally occur to those skilled in the art are to be construed as being within the scope of the present invention.
It will be understood by those skilled in the art that the summary of the invention provided herein is exemplary and explanatory of the invention and are not intended to be restrictive thereof. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. The composition, methods, and examples provided herein are only illustrative and not intended to be limiting.
The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such a process or method. Similarly, one or more steps of method or components preceded by "comprises... a" does not, without more constraints, preclude the existence of other, steps or components. Appearances of the phrase "in a preferred embodiment", "in an embodiment", "in another embodiment" and similar language throughout this specification may, but not necessarily do, all refer to the same embodiment.
The present invention provides a method and kit for extraction of DNA. More specifically, the present invention provides a method for extraction of DNA from a highly processed sample.

The sample is pre-treated with a pre-treat solvent of desired polarity under specific conditions and then solvent is removed. The pre-treated sample (P) is stored at 4°C. The pre-treat solvent is selected from non-polar solvent such as hexane, acetone or toluene. The preferred solvent used in the process is hexane as hexane is safe for edible purpose.
The purified DNA is extracted by the precipitation method. The DNA is extracted from said pre-treated sample (P) by using an extraction buffer. The extraction buffer is Tris-HCl of pH 8. The pre-treated sample is mixed uniformly with the extraction buffer to obtain a uniform solution (SI). The solution (SI) is then centrifuged under pre-defined centrifugation conditions (CI). The aqueous phase (Al) of centrifuged solution is again centrifuged under pre-defined centrifugation conditions (C2). The aqueous layer of sample (A2) obtained after centrifugation (C2) is mixed with chloroform and vortexed till the solution (S2) turns milky.
The solution (S2) is centrifuged under pre-defined centrifugation conditions (C3). The upper aqueous phase (A3) obtained from C3 is transferred to a tube. Specific amount of isopropanol and glycogen of specific concentrations are added to the tube containing the upper aqueous phase (A3) and mixed properly to obtain uniform solution (S3). The tube containing S3 is incubated under specific incubation conditions (II) to obtain incubated solution (S4).
The incubated solution (S4) is centrifuged under pre-defined centrifugation conditions (C4) to obtain pellet (PI). The isopropanol is removed and the pellet is air dried. The air dried pellet is washed with washing solution. The washing solution is ethanol of specific concentration. The solution of pellet and washing solution (S5) is centrifuged under pre-defined centrifugation conditions (C5) to obtain pellet (P2). The washing solution is removed and the pellet (P2) is air dried. The air dried

purified pellet (P2) is dissolved in a elution buffer/ buffer solution and stored. The buffer solution is Tris-EDTA buffer (TE).
Additional purification step is carried out when no proper pellet or coloured pellet (PI) are obtained after the extraction through precipitation. The purification step comprises of mixing of pellet (P2) with chloroform and vortexing till the solution turns milky, referred to as (S2) and undergoes repetition of all the steps of extraction till purified pellets (P2) is obtained.
As the efficiency of amplification process is significantly dependent upon the quantity, quality and purity of the isolated DNA, the quantity and quality of extracted DNA is assessed through Nanodrop Spectrophotometer and Agarose gel electrophoresis. The extracted purified DNA is amplified by PCR amplification. The PCR amplification is done by thermal cycler to increase the amount of DNA using actin gene as the internal standard gene. Actin gene was selected as the housekeeping gene because it is highly conserved and ubiquitously expressed in all the eukaryotic cells. The PCR products are checked by agarose gel electrophoresis and faint bands were obtained due to very less amount of DNA available for amplification. The test sample gave positive expression for the actin gene. Positive PCR amplification has been obtained for the DNA even after no bands are obtained on the agarose gel electrophoresis. It is because only one target copy of the gene is required for DNA amplification.
The process of the present invention is simple, user friendly and economic process for extraction of purified DNA from small amount of a highly processed sample. The invention uses only 10 mM Tris HC1, thereby making process economical as compared to other methods in the existing state of the art. In this process, detergents like SDS, CTAB etc. are not used. This process does not utilize salts which bind to DNA. This process is useful for DNA extraction from the samples where DNA is

present in the scattered form (not entrapped inside the cell). This condition occurs in highly processed samples where only traces of DNA are left in the sample due to breaking of cells containing DNA.
The present invention also provides a kit for extracting DNA from highly processed samples and subsequent amplification of the extracted DNA. The kit of the present invention is economic, simple, highly sensitive and user friendly.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1 depicts flow chart of the process of present invention.
Figure 2 depicts assessment results of DNA through Agarose gel electrophoresis.
Figure 3 depicts DNA amplification results by using Thermal cycler.
DETAILED DESCRIPTION OF THE INVENTION WITH NON-LIMITING EMBODIMENTS AND ILLUSTRATIONS
The financial support to the development of this invention was jointly provided by Department of Science and Technology-Science and Engineering Research Board (DST-SERB), New Delhi; B.L. Agro Industries Ltd., Bareilly, Uttar Pradesh, India; Confederation of Indian Industry (CII), New Delhi under the Prime Minister's Fellowship Scheme for Doctoral Research and Banasthali Vidyapith. Further, the research facilities provided by Prof. Aditya Shastri, Vice-Chancellor, Banasthali Vidyapith are also gratefully acknowledged.
The chemicals used in the invention were procured from Himedia, Invitrogen, Thermoscientific and Fermentas. The reagents and solvents used in the invention are of analytical grade.
The present invention provides a novel process for extraction of DNA. The process comprises isolation of purified DNA from a sample. The DNA obtained after

precipitation is if not pure, additional purification process is carried out. The minute quantities of purified DNA are amplified.
The process of the present invention is outlined in the Figure 1. The sample is pre-treated with a pre-treat solvent of desired polarity under specific conditions and then solvent is removed. The pre-treated sample (P) is stored at 4°C. The solvent is a non-polar solvent selected from hexane or acetone or toluene. The preferred pre-treat solvent is hexane.
The purified DNA is extracted by the precipitation. The DNA is extracted from said pre-treated sample (P) by using an extraction buffer. The extraction buffer is Tris-HCl ofpH8.
Tris based buffer is very efficient in maintaining the pH of the system because the pKa value of Tris HCl is 8.1. This helps in maintaining the pH of the solution within the range of 7 to 9. The process is pH and temperature sensitive. The role of Tris HCl is mainly reported as a buffer in the lysis buffer for DNA isolation. The use of solely Tris HCl for DNA extraction from small amount of sample has not been reported so far. The inventors of the present invention strategically experimented on the finding that Tris HCl most probably interacts with the lipopolysaccharides present in the cell membrane which leads to disintegration of the cell membrane. Apart from its novel role in solely isolating DNA it also stabilizes small amount of DNA isolated either in the integrated or disintegrated form. Different concentrations of Tris HCl are used for DNA extraction. Among various concentrations of Tris HCl used for DNA extraction, the workable range found is 10-20 mM, while the optimum results are obtained at 10 mM concentration of Tris HCl.
The pre-treated sample is mixed uniformly with the extraction buffer to obtain a uniform solution (SI). The solution (SI) is then centrifuged under pre-defined

centrifugation conditions (CI). The aqueous phase (Al) of centrifuged solution is again centrifuged under pre-defined centrifugation conditions (C2). The aqueous layer of sample (A2) obtained after centrifugation (C2) is mixed with chloroform and vortexed till the solution (S2) turns milky.
The solution (S2) is centrifuged under pre-defined centrifugation conditions (C3). The upper aqueous phase (A3) obtained from C3 is transferred to a tube. Specific amount of isopropanol and glycogen of specific concentrations are added to the tube containing the upper aqueous phase (A3) and mixed properly to obtain uniform solution (S3). The tube containing S3 is incubated under specific incubation conditions (II) to obtain incubated solution (S4).
The incubated solution (S4) is centrifuged under pre-defined centrifugation conditions (C4) to obtain pellet (PI). The isopropanol is removed and the pellet is air dried. The air dried pellet is washed with washing solution. The washing solution is ethanol of specific concentration. The solution of pellet and washing solution (S5) is centrifuged under pre-defined centrifugation conditions (C5) to obtain pellet (P2). The washing solution is removed and the pellet (P2) is air dried. The air dried purified pellet (P2) is dissolved in a buffer solution and stored. The buffer solution is preferably Tris-EDTA buffer (TE).
Glycogen is used to precipitate very small amount of DNA. Glycogen is not soluble in ethanol and isopropanol, and therefore, it forms a precipitate which entraps the DNA. After the process of centrifugation, the glycogen-nucleic acid complex forms a visible precipitate on the sides of the tube, which can be separated by further processing, wherein further processing includes the steps of washing of pellet with a washing buffer and further centrifugation step. The glycogen acts as an inert carrier, and therefore, do not interact chemically with the isolated DNA. It precipitates DNA with greater efficiency from diluted solutions.

Additional purification step is carried out when no proper pellet or coloured pellet (PI) are obtained after the completion of extraction process. This can be due to the presence of high polyphenols or protein content. The purification step comprises of mixing of pellet (P2) with chloroform and vortexing till the solution turns milky, referred to as (S2) and undergoes repetition of all the steps of extraction till purified pellets (P2) is obtained.
As the efficiency of amplification process is significantly dependent upon the quantity, quality and purity of the isolated DNA, the quantity and quality of extracted DNA is assessed through Nanodrop Spectrophotometer and Agarose gel electrophoresis. The extracted purified DNA is amplified by PCR amplification. The PCR amplification is done by thermal cycler to increase the amount of DNA using actin gene as the internal standard gene. Actin gene was selected as the housekeeping gene because it is highly conserved and ubiquitously expressed in all the eukaryotic cells. The PCR products are checked by agarose gel electrophoresis and faint bands were obtained due to very less amount of DNA available for amplification. No DNA bands are obtained on 1.2% agarose gel after the process of DNA extraction due to the degradation and shearing of DNA during the industrial process. In case of DNA isolation from highly processed samples agarose gel electrophoresis based results are not the final indication of DNA isolation process. The test sample gave positive expression for the actin gene. Positive PCR amplification has been obtained for the DNA even after no bands are obtained on the agarose gel electrophoresis. It is because only one target copy of the gene is required for DNA amplification.
The process of the present invention is simple, user friendly and economic process for extraction of purified DNA from small amount of a highly processed sample. The invention uses only 10 mM Tris HC1, thereby making process economical as compared to other methods in the existing state of the art. In this process, detergents like SDS, CTAB etc. are not used. This process does not utilize salts which bind to

DNA. This process is useful for DNA extraction from the samples where DNA is present in the scattered form (not entrapped inside the cell). This condition occurs in highly processed samples where only traces of DNA are left in the sample due to breaking of cells containing DNA.
The present invention also provides a kit for extracting DNA from highly processed samples and subsequent amplification of the extracted DNA. The kit of the present invention is economic, simple, highly sensitive and user friendly.
In a preferred embodiment of the present invention, the DNA is isolated from sample of lecithin. Lecithin products are mainly obtained during refining of the vegetable oils (soyabean, rice bran, sunflower, and rapeseed). It is obtained as a gummy substance during the degumming step. Degumming of vegetable oils is done to remove the phosphatides present in the form of gums and also to improve the stability of oil for further processing. Lecithin products of animal origin are obtained from eggs and milk.
PROCESSING OF SAMPLE
The lecithin sample has been pre-treated with hexane to remove different impurities present in lecithin sample.
PRECIPITATION STEP
In the next step, lecithin free from impurities obtained after processing of sample has been mixed with different concentrations of Tris HC1, said concentrations being 5 mM, 10 mM, 45 mM, 50 mM and 100 mM.
The sample has been vortexed vigorously with the extraction buffer for 90 minutes at room temperature to obtain uniform solution (Sri). The solution (Sri) has been then centrifuged at 13000 rpm for 25 minutes at room temperature (RT) (Crl). The

aqueous phase (Arl) has been then transferred in 2 ml centrifugation tube and again centrifuged at 13000 rpm for 25 minutes at RT (Cr2). 1 ml. of the aqueous layer (Ar2) obtained after centrifugation (Cr2) has been transferred in 1.5 ml centrifugation tube and 500 ul chloroform has been added. The solution has been vortexed vigorously for 2-3 minutes till the solution turned milky (Sr2). The solution (Sr2) has been centrifuged at 13000 rpm for 15 minutes at RT (Cr3). The 700 ul of upper aqueous phase (Ar3) has been transferred to 1.5 ml centrifugation tube. 0.8 volumes of 560 ul isopropanol and 4 ul glycogen (3 mg/ml) has been added and gentle mixing was done by inverting the tube for 4-5 times to obtain uniformly mixed solution (Sr3). The tube containing Sr3 has been incubated at room temperature for 30 minutes (Irl) to obtain incubated solution (Sr4). After incubation, the tube containing incubated solution (Sr4) has been centrifuged at 14000 rpm for 20 minutes at 25°C (Cr4) to obtain pellet (Prl). The pellet (Prl) has been air dried to remove all the droplets of isopropanol. The pellet (Prl) has been washed with 70% ethanol (500 ul) (Sr5) and then centrifuged at 14000 rpm for 20 minutes at 25°C (Cr5) to obtain pellet (Pr2). The ethanol has been pipetted out without disturbing the pellet (Pr2). The pellet (Pr2) has been air dried and dissolved in TE (Tris-EDTA) buffer (25 ul) and stored at -20°C.
Examples:
Traces of DNA are present in rice bran lecithin as it is a highly processed material obtained during the refining of the rice bran oil.
Example 1: Methods and conditions for DNA extraction from rice bran lecithin from 200 mg rice bran lecithin as test sample
10 gram of crude rice bran lecithin has been dissolved in 100 ml of hexane as solvent and centrifuged for 10 minutes at 7000 rpm. Desolventization has been carried out by rotary evaporator to separate the solvent from lecithin and was stored at 4°C.

DNA has been extracted from 200 mg of rice bran lecithin in 3 ml of extraction buffer (Tris-HCl, pH 8). Different concentrations of Tris HC1 (5 mM, 10 mM, 45 mM, 50 mM and 100 mM) have been used as the extraction buffer. The sample has been vortexed vigorously with the extraction buffer for 90 minutes at room temperature to obtain a uniform solution. The solution has been then centrifuged at 13000 rpm for 25 minutes at 25°C.
The aqueous phase of centrifuged solution has been then transferred in 2 ml centrifugation tube and again centrifuged at 13000 rpm for 25 minutes at 25°C. The aqueous layer (1 ml) has been then transferred in 1.5 ml centrifugation tube and chloroform (500 ul) has been added. The solution has been vortexed vigorously for 2-3 minutes till the solution turned milky. Further, the solution has been centrifuged at 13000 rpm for 15 minutes at 25°C. After centrifugation, the upper aqueous phase (700 ul) has been transferred in a 1.5 ml centrifugation tube. 0.8 volumes of isopropanol (560 ul) and 4 ul glycogen (3 mg/ml) have been added and gentle mixing has been done by inverting the tube for 4-5 times. The tube has been put for incubation at room temperature for 30 minutes. After incubation, the tube has been centrifuged at 14000 rpm for 20 minutes at 25°C. The pellet has been obtained on the side of the tube and isopropanol has been pipetted out carefully without disturbing the pellet. The pellet has been air dried to remove all the droplets of isopropanol. The pellet has been washed with 70% ethanol (500 ul) and then centrifuged at 14000 rpm for 20 minutes at 25°C. The ethanol has been pipetted out without disturbing the pellet. The pellet has been air dried and dissolved in TE (Tris-EDTA) buffer (25 ul) and stored at -20°C.
Additional purification step has been carried out when no proper pellet or coloured pellet is obtained after the precipitation step. Chloroform (500 ul) has been added to the tube and is vortexed for 2-3 minutes till the solution turned milky. Further, the solution has been centrifuged at 13000 rpm for 15 minutes at 25°C. After

centrifugation, the upper aqueous phase (700 ul) has been transferred in a 1.5 ml tube. 0.8 volumes of isopropanol (560 ul) and 4 ul glycogen (3 mg/ml) have been added and gentle mixing has been done by inverting the tube 4-5 times. The tube has been incubated at room temperature for 30 minutes. After incubation, the tube has been centrifuged at 14000 rpm for 20 minutes at 25°C. The pellet has been obtained on the side of the tube and isopropanol has been pipetted out carefully without disturbing the pellet. The pellet has been air dried to remove all the droplets of isopropanol. The pellet has been washed with 70% ethanol (500 ul) and then centrifuged at 14000 rpm for 20 minutes at 25°C. The ethanol has been pipetted out without disturbing the pellet. The pellet has been air dried and dissolved in TE (Tris-EDTA) buffer (25 ul) and stored at -20°C.
The quantity and quality of DNA has been assessed through Nanodrop Spectrophotometer and Agarose gel electrophoresis and results have been shown in Table 1 and Figure 2. No bands have been obtained on 1.2 % agarose gel.
Figure 2 shows no DNA bands on 1.2% agarose gel. M is the marker (100 bp), lane 1 represents DNA isolated at 5 mM Tris HCl from 200 mg lecithin, lane 2 represents DNA isolated at 5 mM Tris HCl from 1 g lecithin, lane 3 represents DNA isolated at 10 mM Tris HCl from 200 mg rice bran lecithin, lane 4 represents DNA isolated at 10 mM Tris HCl from 1 g rice bran lecithin and lane 5 represents DNA isolated at 45 mM Tris HCl from 200 mg rice bran lecithin, lane 6 represents DNA isolated at 45 mM Tris HCl from 1 g rice bran lecithin, lane 7 represents DNA isolated from 50 mM Tris HCl from 200 mg rice bran lecithin, lane 8 represents DNA isolated from 50 mM Tris HCl from 1 g rice bran lecithin, lane 9 represents DNA isolated from 100 mM Tris HCl from 200 mg rice bran lecithin and lane 10 represents DNA isolated from 100 mM Tris HCl from 1 g rice bran lecithin.

Example 2: Methods and conditions for DNA extraction from rice bran lecithin from 1 gram rice bran lecithin as test sample
DNA has been extracted from 1 gm of rice bran lecithin in 3 ml of extraction buffer (Tris-HCl, pH 8). Different concentrations of Tris HC1 (5 mM, 10 mM, 45 mM, 50 mM and 100 mM) have been used as the extraction buffer. The sample has been vortexed vigorously with the extraction buffer for 90 minutes at room temperature. The solution has been then centrifuged at 13000 rpm for 25 minutes at 25°C. The aqueous phase has been then transferred in 2 ml centrifugation tube and again centrifuged at 13000 rpm for 25 minutes at 25°C. The aqueous layer (1 ml) has been then transferred in 1.5 ml centrifugation tube and chloroform (500 ul) has been added. The solution has been vortexed vigorously for 2-3 minutes till the solution turned milky. Further, the solution has been centrifuged at 13000 rpm for 15 minutes at 25°C. After centrifugation, the upper aqueous phase (700 ul) has been transferred in a 1.5 ml tube. 0.8 volumes of isopropanol (560 ul) and 4 ul glycogen (3 mg/ml) have been added and gentle mixing has been done by inverting the tube 4-5 times. The tube was incubated at room temperature for 30 minutes. After incubation, the tube was centrifuged at 14000 rpm for 20 minutes at 25°C. The pellet has been obtained on the side of the tube. Isopropanol has been pipetted out carefully without disturbing the pellet. The pellet has been air dried to remove all the droplets of isopropanol. The pellet has been washed with 70% ethanol (500 ul) and then at 14000 rpm for 20 minutes at 25°C. The ethanol has been pipetted out without disturbing the pellet. The pellet has been air dried and dissolved in TE (Tris-EDTA) (25 ul) and stored at -20°C.
Additional purification step has been carried out when no proper pellet or coloured pellet has been obtained after the precipitation step. Chloroform (500 ul) has been added to the tube and vortexed for 2-3 minutes till the solution turned milky. Further, the solution has been centrifuged at 13000 rpm for 15 minutes at 25°C. After centrifugation, the upper aqueous phase (700 ul) has been transferred in a

1.5 ml tube. 0.8 volumes of isopropanol (560 ul) and 4 ul glycogen (3 mg/ml) have been added and gentle mixing has been done by inverting the tube 4-5 times. The tube has been incubated at room temperature for 30 minutes. After incubation, the tube has been centrifuged at 14000 rpm for 20 minutes at 25°C. The pellet has been obtained on the side of the tube and isopropanol has been pipetted out carefully without disturbing the pellet. The pellet has been air dried to remove all the droplets of isopropanol. The pellet has been washed with 70% ethanol (500 ul) at 14000 rpm for 20 minutes at 25°C. The ethanol has been pipetted out without disturbing the pellet. The pellet has been air dried and dissolved in TE (Tris-EDTA) (25 ul) and stored at -20°C.
At different concentrations (5 mM, 45 mM, 50 mM and 100 mM Tris HCl) DNA has not been isolated as no reading was recorded on the nanodrop spectrophotometer. No bands have been obtained in the lanes 1-10 on the gel as shown in Figure 2. No bands have been obtained on 1.2 % agarose gel for the DNA extracted by 10 mM Tris HCl as shown in Figure 2.
DNA has been extracted at 10 mM Tris HCl concentration from both the amounts of test sample (200 mg and 1 gm). The quantity of DNA isolated from 10 mM Tris HCl was being assessed through Nanodrop and results are shown in Table 1.
TABLE 1: Results of DNA quantification from different quantities of rice bran lecithin as the test sample by using nanodrop spectrophotometer.

260
280
230
260/280
260/230
Concentration (ng/ ul) 1.8

200 mg 1.0 g test sample
test sample (lecithin) (lecithin)
0.036 0.117
0.048 0.085
0.045 0.170
0.76 1.38
0.81 0.69
1.8 5.9

Example 3: Methods and conditions for DNA amplification
Amplification of DNA through Quantitative (q) PCR (Thermocycler). No amplification has been obtained at concentrations (5 mM, 45 mM, 50 mM and 100 mM) even after the PCR process as bands have not been visualized on the gel even after amplification.
The amplification process for the DNA isolated 10 mM Tris HC1 has been done. The amount of reagent and sample required for the amplification process are shown in Table 2. This method contained Actin as the internal gene. 0.2 ml PCR tube has been taken and the reagents shown in Table 2 have been added. TABLE 2: Amount of sample and reagents required for PCR amplification process.
Reagents uL
Autoclaved MQ water 36 uL
Template (DNA) 2 uL
Actin Forward primer 1.5 uL
Actin Reverse Primer 1.5 uL
50 mM MgC12 2 uL
1 OX PCR Reaction buffer 5 uL
(-MgC12)
lOmMdNTPMix 1.5 uL
Taq DNA Polymerase 0.5 uL
TOTAL 50 uL
The tube has been placed in the thermal cycler to amplify the DNA using the
conditions given in Table 3:
TABLE 3: Conditions for PCR amplification process
Lid: 105°C; Volume: 50 ul

Steps Temperature Time Cycles
1. 94°C 5 min 1
2. 94°C 30 sec 1
3. 50°C 30 sec 1
4. 72°C 30 sec, repeat step 2 for total of 40 cycles 39

5. 72°C 4 min
6. 4°C oo

1

The DNA amplification results have been shown in Figure 3. M is the marker; lane 1 represents amplified DNA from 200 mg rice bran lecithin, lane 2 represents amplified DNA from 1 g rice bran lecithin.
The present invention also provides a kit for DNA extraction. The kit comprises of: I. Pre-treat II. Solution A
III. Solution B
IV. Solution C
V. Washing solution VI. Buffer solution or Elution buffer
The Pre-Treat comprises of a solvent for pre-treatment of sample. The solvent is selected from a non-polar solvent. The preferred Pre-Treat solvent is hexane as it solubilizes lecithin and is a green solvent suitable for edible purpose. The pre-treatment of sample removes non-polar lipid portion from sample.
Solution A comprises of extraction buffer. The extraction buffer comprises of Tris HCl of pH 8 of concentration range from 10 mM to 20 mM. The preferred concentration of Tris HCl for lecithin and rice bran lecithin is lOmM.
Solution B comprises of chloroform solution. Solution C comprises of Solution CI and Solution C2. Solution CI comprises of isopropanol, Solution C2 comprises of glycogen of concentration of 3 mg/ml.

The washing solution comprises of is 70% ethanol. The buffer solution comprises of Tris-EDTA.
The kit of the present invention is economic and free from any salt of reagents causing interference in the amplification of isolated DNA.

We Claim:

1. A novel process for extraction of DNA, said process comprises the steps of:
(a) pre-treating a sample to remove non-polar substances from the sample to obtain pre-treated sample,
(b) mixing said pre-treated sample with an extraction buffer,
(c) precipitating said mixture of step (b) to obtain pellet of DNA (PI),
(d) washing PI with a washing solution and centrifuging to obtain purified and air dried pellet of DNA (P2),
(e) storing said P2 in a buffer solution/elution buffer at specific temperature,
characterized in that
said pre-treating of step (a) is carried out by a non-polar solvent;
said extraction buffer in step (b) is Tris HC1;
said precipitation step comprises pre-defined steps of reactions with specific
reagents under specific reaction conditions and pre-defined steps of
centrifugation under specific conditions to obtain pellet of DNA (PI),
said process enables extraction of small quantity of DNA from even traces of a highly
processed sample.
2. The process for extraction of DNA as claimed in claim 1, wherein said solvent for pre-treatment of sample is selected from acetone or hexane or toluene.
3. The process for extraction of DNA as claimed in claim 1, wherein said solvent is hexane.
4. The process for extraction of DNA as claimed in claim 1, wherein said precipitation step comprises of:

(a) mixing said pre-treated sample (P) with the extraction buffer at pH 8,
(b) vortexing of said mixture of step (a) vigorously for 90 minutes to obtain a uniform solution (SI),
(c) centrifuging said solution of step (b) (SI) at 13000 rpm for 25 minutes at room temperature (RT) (CI),

(d) transferring aqueous phase of the CI (Al) in 2 ml centrifugation tube and again centrifuging at 13000 rpm for 25 minutes at RT (C2),
(e) transferring aqueous layer (1 ml) of the C2 (A2) in 1.5 ml centrifugation tube and adding chloroform (500 ul) and vortexing vigorously for 2-3 minutes till the solution turns milky to obtain solution S2,
(f) centrifuging the solution S2 at 13000 rpm for 15 minutes at RT (C3),
(g) transferring upper aqueous phase (700 ul) of C3 (A3) to 1.5 ml
centrifugation tube and adding 0.8 volumes of isopropanol (560 ul)
and 4 ul glycogen of 3 mg/ml and gentle mixing by inverting the tube
for 4-5 times to obtain solution S3,
(h) incubating the S3 at room temperature for 30 minutes to obtain
incubated solution S4, (i) centrifuging the tube containing S4 at 14000 rpm for 20 minutes at RT
to obtain pellet (PI), (j) pipetting out isopropanol without disturbing the pellet (PI) and air
drying of the pellet (PI).
5. The process for extraction of DNA as claimed in claim 1, wherein said air dried pellet (PI) is washed with washing buffer and centrifuged at 14000 rpm for 20 minutes at RT to obtain purified pellet (P2), while the washing buffer is removed and the pellet (P2) is air dried to store in the elution buffer.
6. The process for extraction of DNA as claimed in claim 1, wherein said washing buffer is 70% ethanol.
7. The process for extraction of DNA as claimed in claim 1, wherein said elution buffer is Tris EDTA.
8. The process for extraction of DNA as claimed in claim 1, wherein said air dried purified DNA pellet (P2) is dissolved in TE (Tris-EDTA) buffer (25 ul) and stored at -20°C.

9. The process for extraction of DNA as claimed in any of the preceding claims wherein the process is further comprises of purification step if purified pellet is not obtained or is coloured due to the presence of impurities, wherein said purification step comprises of repetition of precipitation steps from 3(e) to 3(j) steps of precipitation followed by washing and storing of air dried and purified DNA in buffer solution.
10. The process for extraction of DNA as claimed in claim 1 wherein said process is highly sensitive, economical and simple.
11. A process of extraction of DNA from lecithin sample, said process comprises the steps of:

(a) pre-treating the lecithin sample with hexane to remove non-polar substances to obtain pre-treated sample (Pr),
(b) desolventization of pre-treated sample (Pr) by removing the hexane,
(c) mixing said pre-treated and desolventized sample with Tris HC1 lOmM,
(d) vortexing of said mixture of step (c) vigorously for 90 minutes to obtain a uniform solution (Sri),
(e) centrifuging said solution of step (d) (Sri) at 13000 rpm for 25 minutes at 25°C (Crl),
(f) transferring aqueous phase of Crl (Arl) in 2 ml centrifugation tube and again centrifuging at 13000 rpm for 25 minutes at 25°C (Cr2),
(g) transferring aqueous layer (1 ml) of Cr2 (Ar2) in 1.5 ml centrifugation tube and adding chloroform (500 ul) and vortexing vigorously for 2-3 minutes till the solution turns milky to obtain solution Sr2,
(h) centrifuging the solution Sr2 at 13000 rpm for 15 minutes at 25°C
(Cr3), (i) transferring upper aqueous phase (700 ul) of Cr3 (Ar3) to 1.5 ml
centrifugation tube and adding 0.8 volumes of isopropanol (560 ul)

and 4 ul glycogen (3 mg/ml) and gentle mixing by inverting the tube
for 4-5 times to obtain solution Sr3, (j) incubating the Sr3 at room temperature for 30 minutes to obtain
incubated solution Sr4, (k) centrifuging the tube containing Sr4 at 14000 rpm for 20 minutes at
25°C to obtain pellet (Prl), (1) pipetting out isopropanol without disturbing the pellet (Prl) and air
drying of pellet (Prl), (m)washing Prl with washing buffer and centrifuged at 14000 rpm for 20
minutes at 25°C to obtain purified pellet (Pr2), while the washing
buffer is removed and the pellet (Pr2) is air dried to store in Tris
EDTA at -20°C.
12. The process for extraction of DNA as claimed in claim 11, wherein said lecithin is rice bran lecithin.
13. A kit for extraction of DNA, said kit comprising:
i. Pre-treat, said pre-treat comprising a solvent for pre-treatment of
sample, said solvent being selected from a non-polar solvent, ii. Solution A comprising an extraction buffer, said extraction buffer comprises of Tris HC1 of pH 8 of concentration range from lOmM to 20 mM, iii. Solution B comprising a chloroform solution,
iv. Solution C comprising Solution CI and C2, wherein Solution CI comprises of isopropanol and solution C2 comprises of glycogen in the concentration of 3 mg/ml, v. Washing solution comprising 70% ethanol. vi. Buffer solution or Elution buffer comprising Tris-EDTA. wherein said kit is economic and free from any salt of reagents causing interference in the amplification of isolated DNA.
14. The kit for DNA extraction as claimed in claim 13, wherein said Pre-treat solvent is selected from hexane or acetone or toluene.
15. The kit for DNA extraction as claimed in claim 13, wherein said Pre-treat solvent is hexane.
16. The kit for DNA extraction as claimed in claim 13 wherein said Tris HCl is of lOmM concentration.