DNA extraction

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

Cell disruption

An organism's DNA can be isolated in a number of ways. Most of the methods begin with a concentration of the cells by centrifugation and a suitable cell disruption of the cell precipitate for the respective group . So z. B. in plant , fungal or bacterial cells, which in comparison to animal cells, mycoplasmas and some archaea species have a cell wall in addition to the cell membrane , usually additional enzymatic (e.g. lysozyme in bacteria or proteinase K for proteolysis ) or mechanical crushing steps ( blender ) respectively. In plasmid preparation from bacteria, alkaline lysis is often used as the chemical cell disruption . After cell disruption, the homogenate is usually clarified by filtration or centrifugation. DNA from mitochondria or chloroplasts can be separated from the DNA of the cell nucleus by cell fractionation . Hirt extraction is used to isolate extrachromosomal DNA such as viral DNA. An RNase digestion can be performed to remove the RNA .

Extractions

DNA is a polar biopolymer with a relatively high molar mass , which is why it precipitates in a non-polar environment due to the reduced hydrate shell and the resulting reduction in its solubility . In addition, because of the deoxyribose phosphate backbone with negative charges proportional to the chain length, DNA is insoluble in acidic, aqueous solutions. At low pH values , the phosphate groups and thus the negative charges of the DNA are saturated with protons , which also reduces the size of the hydration shell.

Most DNA extractions are based on two different methods after cell disruption , two- phase extraction or precipitation , the latter possibly with additional selective adsorption onto a DNA-binding matrix. The extraction processes are also sometimes combined with one another. The following methods are usually followed by a final ethanol precipitation.

Two-phase extraction

RNA extraction by two-phase extraction and ethanol precipitation.

The two-phase extraction is based on the different distribution of biomolecules in an organic phase, an aqueous phase and the interphase in between. These include B. the phenol-chloroform extraction , the phenol-chloroform- isoamyl alcohol extraction and the Trizol extraction. During extraction according to Sacchi (with Trizol , TRI Reagent , Trisure , STAT-60 , RNAzol or DNAzol as trade names), the DNA collects in the interphase, the RNA and the carbohydrates in the aqueous phase and the proteins and the lipids in the organic Phase. Here that provides chaotrope guanidine thiocyanate for denaturation , all proteins including DNase , RNase and peptidases . The interphase is transferred to a new vessel with a pipette . The DNA contained is then subjected to ethanol or isopropanol precipitation .

Adsorption on silica gel

Commercially available silica gel column, upside down

In this method the DNA is precipitated in slightly acidic environment to a matrix of silica gel ( English silica gel ), glass fiber filters or diethyl aminoethyl-dextran ( DEAE-dextran ) adsorbed . Adsorption takes place under DNA-precipitating buffer conditions (with alcohols and a slightly acidic pH value) with silica and glass fiber via polar interactions, with DEAE-dextran via ionic interactions. The sample can be applied several times to the matrix in order to increase the yield . The matrix contained in the chromatography column is then freed from the contaminating DNA-binding proteins using chaotropes. The chaotropic salts denature the proteins and keep them in solution while the DNA remains bound to the matrix. In a chemical gel extraction , i.e. the extraction of DNA from agarose gels by dissolving the gel, sodium iodide is often used as an alternative chaotrope . The DNA is eluted from the matrix by adding water or slightly basic Tris buffer with EDTA ( TE buffer ). An optional second elution increases the absolute amount of DNA extracted at the expense of the concentration. After the DNA has been eluted from the silica gel matrix, ethanol is usually also precipitated.

Lysis by boiling

In boiling lysis , bacterial cells are boiled for 45 seconds a short time after the addition of lysozyme and Triton X-100 , after which the clumped cell debris (with the majority of the chromosomal DNA) is removed with a sterile toothpick . After centrifugation, the now cleared supernatant is subjected to ethanol precipitation. This method is mostly used to isolate plasmids from bacteria, since the chromosomal DNA clumps together with other components. This method is suitable for a higher number of samples, but produces a comparatively somewhat lower yield and purity, since plasmids are partially removed with the clumped components and the proteins are not removed selectively.

CTAB precipitation

RNA and DNA form insoluble complexes with cationic surfactants . CTAB precipitation uses the extraction of DNA with cetyltrimethylammonium bromide (CTAB), which is then combined with chloroform extraction and ethanol precipitation. It is mostly used on plant cells. According to a crushing in a mortar the cell disruption is carried out by addition of CTAB, polyvinyl pyrrolidone and mercaptoethanol in a TRIS - buffer at a pH value of 8. Subsequently, usually a chloroform-isoamyl alcohol or chloroform- octanol extraction, in which the DNA due to of the basic pH remains in the aqueous phase. The aqueous phase is subjected to ethanol precipitation in a new reaction vessel. The extraction with Catrimox-14 , a method of RNA extraction, is carried out analogously .

Ethanol precipitation

Ethanol precipitation of chromosomal DNA from zucchini

In ethanol or isopropanol precipitation, the DNA is precipitated under slightly acidic conditions (pH 5.2) in a less polar (alcoholic) environment due to the reduction in solubility . Water has a dielectric constant of 80.1 at 25 ° C, while that of ethanol is around 24.3. The addition of ethanol removes water from the DNA, which reduces its solubility. The low pH value in the case of ethanol precipitation is achieved by adding an acidic potassium acetate or sodium acetate solution. This keeps the otherwise negatively charged DNA in a partially protonated, partially saturated and uncharged state with potassium or sodium ions, whereby the solubility is further reduced. In the case of an alternative use of ammonium acetate , the proteins can be precipitated separately before the addition of ethanol and separated off by an additional centrifugation step, which allows plasmid DNA to be extracted without using silica.

After adding the ethanol and the potassium acetate solution, the test batch is for 15 min. centrifuged at 10,000 g. At low DNA concentrations or short DNA fragments below 100 bases or base pairs, the precipitation period can be extended to several hours and polymers such as. B. glycogen , tRNA or linear polyacrylamide can be added. The supernatant is removed after centrifugation and discarded. In most cases, 70% ethanol is then added as a washing solution, centrifuged again and the supernatant discarded again. The precipitate is dried and then dissolved in TE buffer. Since the ethanol precipitation separates relatively uncleanly and some proteins, polysaccharides and RNA precipitate at the same time with the DNA, it is usually only used as a final purification step and several times in succession on the same sample.

Endotoxin Removal

In some cases, contaminating endotoxins must be removed from the DNA , e.g. B. to avoid activation of TLR-4 when using purified plasmid DNA in TLR4-positive organisms. For this purpose, two-phase cloud point extraction with Triton-X114 or chromatography are used, both methods in succession or in combination.

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

The material used in a chromatography column for endotoxin removal is, inter alia, hydroxyapatite , polystyrene , Dowex 1-X2 (strongly basic anion exchanger ), activated carbon or polymyxin-B -modified column material.

quantification

The easiest way to determine the amount of purified DNA is by photometry . On the other hand, if the amount of a certain DNA sequence in a mixture of DNA sequences is to be determined, QPCR is usually used. In photometry, an extinction E of 1 of a purified DNA solution for double-stranded DNA and a wavelength of 260 nm corresponds to a concentration of 50 micrograms per milliliter, for single-stranded RNA this corresponds to 40 micrograms per milliliter and for single-stranded DNA 33 micrograms per milliliter. In the case of single-stranded oligonucleotides , the corresponding concentration can be calculated from the number of different nucleic bases ( n A for the number of adenines ):

The extinction coefficient can be calculated for single-stranded and double-stranded DNA using the nearest-neighbor heuristic . An extinction coefficient allows the concentration to be determined without a standard series according to Lambert-Beer’s law . The extinction coefficient ϵ at a wavelength of 260 nm is calculated as follows:

purity

The ratio of the absorbance at 260 nm and the absorbance at 280 nm is used as an indicator of the purity of the DNA. Proteins absorb relatively strongly at 280 nm due to the amino acids they contain phenylalanine , tyrosine and tryptophan . Pure DNA has an absorbance ratio of two, whereas a pure protein sample is 0.57. However, since the extinction coefficient of proteins is more than a power of ten lower than that of DNA, a ratio of 1.9 means a protein content of 40% and a ratio of 1.8 means a protein content of 60%. A calculation of the DNA concentration based on the extinction coefficient is therefore only useful for a ratio of 2. One way of calculating the concentration, including the purity, is based on the following formula, with R as the ratio of the extinctions at 260 nm and 280 nm:

Inserting the extinction coefficients results in:

Where technically available, it makes more sense to use the ratio of the absorbances at 260 nm and 234 nm as a purity indicator. At 234 nm, DNA absorbs little compared to 280 nm, but the peptide bond of the proteins is relatively strong. Since the extinction coefficients for protein at 234 nm are only 1.5 to 1.8 times lower than those of DNA, this measure is more sensitive to protein contamination. Similarly, the concentration can be calculated taking the purity into account using the following formula:

history

From 1951 E. Volkin and CE Carter first used the chaotrope guanidinium chloride for denaturing proteins with subsequent chloroform extraction of the RNA and ethanol precipitation, from 1963 it was used by R. Cox and H. Arnstein for RNA extraction. The two-phase extraction, on which the three current two-phase extraction methods are based, comes from an extraction method with methanol and chloroform, which was developed in 1959 for lipids. Phenol was first used in 1953 by W. Grassman and G. Deffner for denaturing protein extraction, from 1956 phenol was used by K. Kirby for RNA extraction. The use of the chaotrope guanidinium thiocyanate was first mentioned in 1977 by A. Ullrich and described in 1979 by J. Chirgwin. From 1979, H. Birnboim and J. Doly combined the alkaline lysis of bacterial cells with DNA extraction. In 1981 J. Feramisco described an RNA extraction with guanidinium thiocyanate and hot phenol. In 1985, J. Zeugin and J. Hartley published the current variant of ethanol precipitation. In 1987, Piotr Chomczynski and Nicoletta Sacchi first used guanidinium thiocyanate in combination with two-phase extraction for denaturing and solubilizing proteins at acidic pH values ​​during extraction and precipitation of the DNA. In 1990 the selective adsorption of DNA on silica gel was published by R. Boom.

literature

Individual evidence

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