Nucleic acid methods
DNA purification (including DNA preparation, DNA isolation ) describes the separation of DNA from a mixture or a solution containing a plurality of biomolecules.
Principle
DNA has negative charges due to the Phosphoriboserückgrates proportional to their chain length and is insoluble due to its relatively high molecular weight in an acidic aqueous environment, as the phosphate groups with proton is saturated and decreases the hydration, and hence the solubility in a row. Likewise, DNA is in a nonpolar environment (organic solvent ) due to the reduced hydration shell and the lower solubility insoluble.
In comparison to intracellular proteins DNA has a significantly higher molecular weight, a higher density, and no positive charges. Because of the conjugated double bonds in the DNA nucleobases absorbed ultraviolet light at a wavelength of 260 nm, which is used for the photometric quantification. Thus, the purification factors can be determined. An absorbance of 1 corresponds to a purified solution of DNA double-stranded DNA at a concentration of 50 micrograms per milliliter, wherein single-stranded DNA or RNA, this corresponds to 40 micrograms per milliliter, and with single-stranded oligonucleotides of 20 micrograms per milliliter. DNA can be visualized by different staining methods. Dyes and procedures eg methylene blue stains all or silver staining can be used. Fluorescent dyes are, for example, 4 ',6- diamidine -2-phenylindole, Bisbenzimide Hoechst 33342, or as phenanthridines, such as acridine orange, ethidium bromide, propidium iodide, gel or gel Red Green. Other DNA-binding molecules such as spermine, spermidine, polyethyleneimine, pentamidine and lexitropsins and DNA-binding proteins.
The purification of DNA may be achieved by various methods can also be combined. The DNA purification can be done by applying different cleaning methods whose effectiveness ( the meaningful sequence ) and their efficiency ( the degree of purification ), followed by analytical methods and quantified. The choice of methods is differentiated according to the chain length between genomic DNA, plastid DNA, plasmids and viral DNA.
Tissues are occasionally crushed against a mechanical cell disruption ( blender ) or enzymatic ( proteinase K). In electrophoresis and chromatography, a sample must first be filtered or centrifuged after cell disruption, since the apparatus can be blocked by the coarse fragments. To inactivate nucleases EDTA or other chelators is usually added and worked quickly at 4 ° C.
Separation processes
DNA - extraction
A series of extractions and precipitations is probably the most widely used method.
Chromatography
DNA can be separated by size exclusion chromatography ( SEC) according to their hydrodynamic volume. Likewise, DNA can be separated by anion exchange chromatography.
Sedimentation
By density gradient centrifugation in a cesium chloride gradient DNA can be separated due to their sedimentation.
By pull-down assays, such as chromatin immunoprecipitation DNA molecules are adsorbed on the basis of their affinity to a matrix and isolated from the properties of the matrix.
Electrophoresis
The DNA may be separated according to their electric charge and their hydrodynamic volume in accordance with another preceding purification by agarose gel electrophoresis or by capillary electrophoresis, both of which depend on the chain length and thus the molecular weight.
Filtration
In a series of microfiltration and ultrafiltration several samples are also separated according to their hydrodynamic volume.