History and introduction of DNA extraction:
DNA extraction is a technique to separate DNA from cell membranes, proteins, and other biological components from a sample using physical and/or chemical processes. For the first instance, DNA isolation was done by Friedrich Miescher in 1869.
The employment of a DNA isolation process should result in an effective extraction of DNA that is clean, abundant, and free of impurities like RNA and proteins. For DNA extraction, both manual techniques and commercially available kits are employed. DNA can be extracted from a variety of tissues, such as blood, bodily fluids, direct fine needle aspiration cytology (FNAC) aspirate, formalin-fixed paraffin-embedded tissues, frozen tissue sections, etc.
Lysing the cells and solubilizing the DNA is the first step in extracting DNA. Chemical or enzymatic techniques are then used to remove macromolecules, lipids, RNA, or proteins. Techniques for extracting DNA include salting out and proteinase K treatment, chemical extraction (phenol-chloroform method), and adsorption method (silica–gel membrane).
Principle of DNA Isolation:
For all DNA manipulation experiments, high-quality DNA is a need. Disrupting the cell wall and cell membrane is the primary step in all plant DNA extraction procedures. The nuclear membrane allows DNA to be released into solution, followed by precipitation while contaminating biomolecules such as proteins, polysaccharides, lipids, phenols, and other secondary metabolites are cleared away.
This is accomplished by breaking up the tissue in a mortar and pestle with the use of liquid nitrogen, and the various elements of the homogenization or extraction buffer are then added, along with the appropriate precipitating and purification techniques.
Since DNA can be extracted from a variety of tissues, including seedlings, leaves, cotyledons, seeds, endosperm, tissue culture callus, roots, etc., the approach of DNA extraction to be used by the experimenter depends on the tissue type and the ultimate concentration of DNA required.
What are the components which involve DNA Isolation?
A buffer for extraction:
This includes a chelating agent like EDTA which chelates the magnesium ions needed for DNase activity, a detergent-like cetyl trimethyl ammonium bromide (CTAB) or SDS which disrupts the membranes, a reducing agent like B mercaptoethanol which helps in denaturing proteins by breaking the disulfide bonds between the cysteine residues and for removing the tannins and polyphenols present in the crude extract, and a buffer Tris with a pH of 8 and a salt, such as sodium chloride, help precipitate by balancing the DNA’s negative charges and allowing the molecules to bind.
Using chloroform to extract phenol:
Unwanted contaminants, mostly proteins, are a typical component of nucleic acid solutions. The nucleic acid solution is progressively washed with a volume of phenol (pH 8.0), a volume of phenol: chloroform: isoamyl alcohol (25: 24: 21), and a volume of chloroform: isoamyl alcohol to extract the nucleic acid ( 24:1). Intermittent centrifugation is used to move the upper aqueous phase to a fresh tube without contacting the interphase.
The nucleic acids are kept in the aqueous phase while the contaminants denature and collect in the organic phase or in the thin layer that separates the two phases. Using the enzyme proteinase K, which is once more denatured by phenol via phenol-chloroform extraction, is another method of eliminating proteins.
Nucleic acid precipitation:
The most popular technique for nucleic acid precipitation is alcohol precipitation. The nucleic acid must first be diluted with a monovalent salt before being mixed gently with alcohol. By centrifuging, the nucleic acid that spontaneously precipitates can be pelletized. Washing with 70% alcohol gets rid of the salt and alcohol residue. Sodium acetate pH 5.2 (final volume 0.3M), sodium chloride (final concentration 0.2M), ammonium acetate (2-2.5M), lithium chloride (0.8M), and potassium chloride are the most often utilized salts. The usual alcohols used for nucleic acid precipitation are ethanol (two times the volume) or isopropanol (two-thirds the volume).
Resuspending DNA:
The nucleic acid pellet can be resuspended in either sterile distilled water or TE(10 \smM Tris:1mM EDTA)
DNA purification:
The nucleic acid solution is incubated with RNase A (10 mg/ml) at 37° C, and then the DNA is purified by reprecipitation after phenol: chloroform extraction to eliminate the RNase.
What are the conventional methods of DNA isolation?
Reagents;
1. The CTAB Extraction Buffer
100 mM Tris, 1.4 M Na Cl (pH 8.0)
EDTA 20 mM (pH 8.0)
2-Mercaptoethanol, 2-CTAB
Isopropanol, 2.
3. phenol-saturated pH 8.0
4. A combination of chloroform and isoamyl alcohol (24:1)
5. 10 mM Tris:EDTA (10 mM: 1 mM) pH 8.0
RNase A (10 mg per ml):
RNase A should be dissolved in 10 mM Tris-Cl, 15 mM NaCl, pH 7.5. Heat for 15 minutes at 1000 C. Until room temperature, cool. Aliquots should be kept at -20 C.
- 70% ethanol,
Unspecified:
- Pestle and mortar
- sterilized tips and pipettes
- Microcentrifuge and centrifuge tubes that are sterile
- Sterilized glasses
What is the procedure for DNA Isolation?
- After being frozen in liquid nitrogen, 2 g of fresh, young leaf tissue should be weighed and ground into a fine powder in a pestle and mortar.
- Transfer to a water bath maintained at 65° C with a 50 ml centrifuge tube and 10 ml of extraction buffer. Vortex or aggressively combine.
- Incubate for an hour at 65 °C. Intermittently combine. Let the food reach room temperature.
- Add 10 ml of isoamyl alcohol: chloroform. Gently combine by forming an “8” shape.
- Centrifuge at 25 °C for 10 minutes at 10,000 rpm.
- The aqueous phase is transferred to a brand-new centrifuge tube. Add 0.6 volume of cold isopropanol, and then wait for the DNA to precipitate for 30 minutes in a deep freezer set to – 20° C.
- Spool the DNA out. Using a pipette, drain the surplus chemicals.
- Add 0.5 milliliters of 70% ethanol. Gently combine, then let it sit at room temperature for 15 minutes.
- Repeat the 70% ethanol process after decanting. Remove the liquid and vacuum or air dry the pellet.
- DNA should be dissolved in 1 cc of 10:1 TE.
- Include 10 l of RNAse A, then incubate for an hour at 37 °C.
- Add phenol, chloroform, and isoamyl alcohol in an equal volume (25:24:1), carefully mix for at least five minutes, then centrifuge at 10,000 rpm for ten minutes. Isomalol was extracted twice using chloroform.
- Add 1/10 volume of 3M NaOAc and 2.0 times the total amount of cooled ethanol to precipitate DNA. The DNA can either be precipitated by centrifugation at 12,000 rpm for 15 minutes or by gently mixing and spooling out the mixture. Wash the area twice with 70% ethanol to get rid of excess salt. Dry using a vacuum or by air.
- Include the bare minimum of TE (10:1). Keep the pellet suspended at room temperature. Keep at – 20 ° C.
What are the precautions?
- Material that has been thoroughly ground in liquid nitrogen must be delivered right away to the extraction buffer; it cannot be allowed to “sweat.”
- To guarantee complete DNA recovery during chloroform: isoamyl alcohol extraction, the aqueous phase should be carefully removed and the organic phase should be extracted again. Dilution with more digestion buffer and re-extraction are the solutions if no separation is seen between the two phases, which may be caused by a high concentration of DNA and/or cell debris in the aqueous phase.
- The procedures should be performed with the utmost care. To prevent DNA from being sheared, techniques like vortexing and pipetting with fine tips should be avoided.
- DNA should not be over-dried as TE resuscitation becomes challenging.
- The glassware, plasticware, pestles, mortars, and other items should be thoroughly decontaminated. In order to avoid cross-contamination, caution should be exercised.
- In addition to regular extractions, blank extraction controls are performed to look for contamination.
References:
https://projects.nfstc.org/workshops/resources/literature/Extraction/05
https://en.wikipedia.org/wiki/DNA_extraction
https://www.sciencelearn.org.nz/resources/2036-dna-extraction
