All living things, including animals, plants, and bacteria, have DNA in their cells. DNA is a very long molecule made up of a chain of nucleotides and the order of these nucleotides is what makes organisms similar to others of their species and yet different as individuals.
Genes are sections within this long DNA molecule. In order to study DNA, you first have to get it out of the cell. In eukaryotic cells, such as human and plant cells, DNA is organized as chromosomes in an organelle called the nucleus. Bacterial cells have no nucleus. DNA extraction and polymerase chain reaction PCR are the basic techniques employed in the molecular laboratory. This short overview covers various physical and chemical methods used for DNA extraction so as to obtain a good-quality DNA in sufficient quantity.
The basic principle and different variants of PCR are discussed. Friedrich Miescher in did DNA isolation for the first time. The use of DNA isolation technique should lead to efficient extraction with good quantity and quality of DNA, which is pure and is devoid of contaminants, such as RNA and proteins.
Manual methods as well as commercially available kits are used for DNA extraction. Various tissues including blood, body fluids, direct Fine needle aspiration cytology FNAC aspirate, formalin-fixed paraffin-embedded tissues, frozen tissue section, etc. DNA extraction involves lysing the cells and solubilizing DNA, which is followed by chemical or enzymatic methods to remove macromolecules, lipids, RNA, or proteins.
DNA extraction techniques include organic extraction phenol—chloroform method , nonorganic method salting out and proteinase K treatment , and adsorption method silica—gel membrane. Cell lysis can be done using nonionic detergent sodium dodecyl sulfate , Tris—Cl, and Ethylene diamine tetraacetic acid EDTA , and this step is followed by removal of cell debris by centrifugation.
Protease treatment is then used to denature proteins. Precipitation with ice-cold ethanol is performed for concentrating DNA. Nucleic acid precipitate is formed, when there is moderate concentration of monovalent cations salt.
This precipitate can be recovered by centrifugation and is redissolved in TE buffer or double-distilled water. Spectrophotometry involves estimation of the DNA concentration by measuring the amount of light absorbed by the sample at specific wavelengths. A ration of less than 1.
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