DNA purification is prerequisite step for an array of biomolecular analyses such as PCR, electrophoresis, DNA sequencing, fingerprinting, genetic engineering and cloning. It plays an important role in understanding the genetic cause of disease, detection of virus and bacteria. This technique is essential for many diagnostic tests.
DNA extraction is the process of separating DNA from cells present in any biological sample. The purpose is to separate DNA in the nucleus from other cellular components found in the cells. The extracted DNA should be of good quality and in efficient amount. Quality of purified DNA is determined by purity of DNA without contamination such as RNA, proteins, detergents etc. DNA can be extracted by manual method as well as by commercially available kits.
Step 1. Breaking the cells open to release the DNA
The cells in the sample are lysed by physical or chemical methods in a solution containing salt and detergent. The detergent breaks down the lipids in the cell membrane and nuclei. Hence DNA is released as the cell membranes are disrupted.
Step 2. Separating DNA from proteins and other cellular debris
Often a protease is added to degrade DNA-associated proteins.
Step 3. Precipitating the DNA with an alcohol
Ice-cold ethanol or isopropanol added to the DNA sample. DNA is soluble in water but insoluble in salt and alcohol. So by stirring the alcohol layer with a sterile pipette, the precipitate is visible and can be taken out. If there is lots of DNA, you may see a white precipitate.
Step 4. Cleaning the DNA
The DNA sample can now be further purified. It is then resuspended in a slightly alkaline buffer.
Step 5. Confirming the presence and quality of the DNA
Optical density readings taken by a spectrophotometer can be used to find out the concentration and purity of DNA in a sample. Further, gel electrophoresis can be used to ascertain the presence of DNA in the sample.
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