Basic steps in DNA extraction

DNA isolation is a routine procedure to collect DNA for subsequent molecular or forensic analysis. There are three basic and two optional steps in a DNA extraction

Homogenization/ disruption of cells/ Cell lysis

Breaking the cells open, commonly referred to as cell disruption or cell lysis. Cell walls and membranes must be broken to release the DNA and other intracellular components. This is usually accomplished with an appropriate combination of enzymes to digest the cell wall (usually lysozyme) and detergents to disrupt membranes. This is commonly achieved by chemical, physical and enzymatic

methods.

 I.           Physical disruption

1. Freeze/thawing

Ø Ice dendrites, needles, that can puncture and disrupt the cell membrane,

     thereby releasing the content of the cell in the solvent

2. Grinding in liquid nitrogen

Ø Samples of cells are frozen in liquid nitrogen and subsequently crushed

      (e.g. with the aid of a pestle and a mortar)

3. Ultra-sonication

Ø Ultrasonic waves generate friction forces that might disrupt cell membranes and

      generate shear in large biological molecules.

4. Bead milling

Ø The collision forces generated by shaking beads may disrupt cellmembranes and

      thereby releasing the content of cells

II.        Chemical lysis

1.Ionic detergents

e.g. sodium dodecyl sulphate (SDS),

potassium ethyl

xanthogenate (PEX),

cetyl trimethyl ammonium bromide (CTAB)

Ionic detergents are known to:

• denature proteins

• inhibit enzymes

• interact strongly with lipids.

The direct action of detergents is probably cleaning the cell membrane

of its lipids, which ultimately leads to the destruction of the cytoplasmic

membrane causing the complete lysis of the cell

2. Non-ionic solvents

e.g. butanol

Aim is to dissolve certain structural molecules of the cell membrane,

thereby initiating membrane leaks, which may cause cell lysis

3. Special buffers

e.g. Sucrose-method

Drastic change of osmotic environment around cells yield to leaking cell

membranes, which may lead to cell lysis.

III.     Enzymatic lysis

Lysozyme (= muramidase)

•      Hydrolyzes glycosidic bonds (β(1-4) linkages) in carbohydrate

• Isolated from chicken egg white

Lyticase

•      Mixture of endoglucanase and protease

•      hydrolyzes poly-β(1→3)-glucose

Other enzymes e.g. achromopeptidase, Labiase, Lysostaphin

•      Isolated from different (bacterial) organisms

 Removal of protein (deprotinization) Carbohydrates, RNA etc.

          RNA is usually degraded by the addition of RNase.(often done)The resulting oligoribinucleotides are separated from the high molecular weight (HMW) DNA by exploiting their differential solubilities in non-polar solvents (usually alcohol/water)

            Proteins are subjected to chemical denaturation and/or enzymatic degradation. The most common technique of protein removal involves denaturation and extraction into an organic phase consisting of phenol and chloroform. DNA-protein interactions are disrupted with SDS (Sodium Dodecyl Sulfate,phenol, or broad spectrum proteolytic enzymes as pronase or proteinase K. Alkaline pH and high concentration of salts improve the efficiency of the process.

Proteins are removed by treatment with phenol or chloroform-isoamyl alcohol or phenol chloroform. Proteins can also be removed by salting out proteins by sodium acetate. Trichloromethane (Chloroform) denatures proteins and lipids and makes DNA less soluble in the organic/phenolic phase. 3-methyl-1-butanol (Isoamyl alcohol) acts as anti-foaming agent and separate layers too.

 Another widely used purification technique is to band the DNA in a CsCl density gradient using ultracentrifugation.

Precipitating the DNA with an alcohol

Usually ice-cold ethanol or isopropanol is used. Suitable +1 cationic salts are; ammonium acetate, Lithium chloride ,sodium chloride. Salts of nucleic acids and monovalent cations are almost insoluble in alcohol-water mixtures (precipitate as pellet).Since DNA is insoluble in these alcohols; it will aggregate together, giving a pellet upon centrifugation. This step also removes alcohol-soluble salt. The DNA in the aqueous phase is precipitated with cold (0^oC) ethanol. The precipitate is usually redissolved in buffer and treated with phenol or organic solvent to remove the last traces of protein, followed by reprecipitation with cold ethanol.

For high purity, physical purification methods are used.

1)   Isopycnic centrifugation

2)   Ultra filtration