DNA extraction

DNA extraction describes the extraction of DNA from cells. DNA extraction is a method for DNA purification.

Cell disruption

The DNA of an organism can be isolated in a number of ways. Most processes start with a concentration of the cells by centrifugation and one suitable for the respective group of cell disruption, the cell precipitate. As need, for example, the case of plant, pilzigen or bacterial cells which have a cell wall, in comparison with animal cells, mycoplasma, and some Archaeenarten in addition to the cell membrane, usually additional enzyme (such as lysozyme in bacteria, or proteinase K proteolysis ) or mechanical grinding steps ( the blender ) take place. The plasmid preparation from bacterial alkaline lysis is often used as a chemical cell disruption. After cell disruption is usually followed by a clarification of the homogenate by filtration or centrifugation. DNA from mitochondria or chloroplasts may be separated by cell fractionation of the DNA of the nucleus.

Extractions

DNA is a biopolymer with a relatively polar, high molecular weight, and therefore it turns in the nonpolar environment of a reduced size, and the consequent lowering of hydration of its solubility. In addition, DNA is proportional to the chain length in acidic aqueous solutions with insoluble negative charges due to the deoxyribose phosphate backbone. At low pH values, the phosphate groups and thus the negative charges of the DNA are satisfied by protons, whereby the hydrate is also reduced.

Most DNA extraction based on the cell information to two different methods, the two-phase extraction and the precipitation reaction, the latter possibly with additional selective adsorption of a DNA binding array. The extraction process can also be combined with each other to some extent. The following methods usually followed by a final ethanol precipitation.

Two - phase extraction

The two- phase extraction is based on the different distribution of biomolecules in an organic phase, an aqueous phase and the intermediate interphase. These include, for example, phenol-chloroform extraction, phenol-chloroform - isoamyl alcohol extraction, and the Trizol extraction. These three methods are based on an extraction method that was developed in 1959 for lipids. The DNA is collected in the extraction according to Sacchi in the interphase, and RNA in the aqueous phase of the carbohydrates and proteins and the lipids in the organic phase. In the case of Trizol, TriReagent, RNAzol or the chaotropic agent guanidinium thiocyanate DNAzol ensures denaturation of all proteins, including DNases, RNases and peptidases. The interphase is transferred with a pipette into a new vessel. Subsequently, the DNA contained an ethanol or isopropanol precipitation is subjected.

Adsorption on silica gel

In this method, the DNA is precipitated in a slightly acidic environment in a matrix of silica gel adsorbs (English silica gel ), glass fiber filters or diethyl aminoethyl- dextran ( DEAE- dextran). The adsorption is taking DNA precipitating buffer conditions ( with alcohols and slightly acidic pH) and in silica fiber over polar interactions with DEAE-dextran by ionic interactions. The sample may be repeatedly applied to the matrix to increase the yield. Subsequently, the matrix contained in the chromatographic column is freed from the contaminating chaotropes DNA-binding proteins. The chaotropic salts denature the proteins and keep them in solution while the DNA remains bound to the matrix. In a chemical gel extraction, thus the extraction of DNA from agarose gels by dissolution of the gel, is often used as an alternative chaotropic sodium iodide. Elution of the DNA from the matrix by addition of water or a slightly basic buffer, Tris EDTA ( TE buffer ). After elution of the DNA from the silica gel matrix usually also follows that ethanol precipitation.

Lysis by boiling

For lysis by boiling (English boiling lysis ) cells in the presence of lysozyme and Triton X-100, boiled 45 seconds, then be with a sterile toothpick clumped cell debris removed ( with the bulk of the chromosomal DNA). After centrifugation, the clarified supernatant then is subjected to ethanol precipitation. This method is usually used for plasmid isolation from bacteria, since the chromosomal DNA clumped with other ingredients. This method is at an elevated number of samples, however, produces a comparatively somewhat lower yield and purity, as no selective removal of proteins takes place.

CTAB precipitation

Another precipitation method uses the selective precipitation of DNA with cetyltrimethylammonium bromide, which is then combined with the ethanol precipitation.

Ethanol precipitation

In ethanol or isopropanol precipitation, the DNA (5.2 pH) precipitated under mildly acidic conditions in a less polar environment due to reduction in solubility. Water has a dielectric constant of 80.1 at 25 ° C, while that of ethanol is about 24.3. The low pH in the ethanol precipitation is achieved by adding acid potassium acetate, or sodium acetate solution. This keeps the otherwise negatively charged DNA in a protonated and uncharged state, whereby the solubility is further reduced. In an alternative use of ammonium acetate, the proteins from the ethanol addition can be made separately, which allows extraction of plasmid DNA without silica. At low DNA concentrations or short DNA fragments less than 100 bases (base pairs ) of the precipitation period can be extended up to several hours and then added as a precipitation aid polymers such as glycogen, tRNA or linear polyacrylamide. As the ethanol precipitation relatively unclean separated and precipitate some proteins, polysaccharides and RNA simultaneously with the DNA, it is usually used only in a row as a final purification step and repeatedly on the same sample.

Endotoxin

In some cases the DNA must be freed from contaminating endotoxins, for example in order to avoid activation of TLR-4 in an application of purified plasmid DNA in organisms. For this purpose, among others, the two-phase cloud point extraction with Triton - X114 or chromatography may be used, both methods sequentially or in combination.

Cloud point extraction is based on phase separation, a one percent ( w / v ) Triton - X114 solution above 22 ° C, or after the addition of salts and sodium lauryl sulfate.

As a material in a chromatography column for endotoxin removal, among other things, hydroxyapatite, polystyrene, Dowex 1 -X2, activated carbon or polymixin B columns -modified material is used.