DNA extraction/isolation is the purification of DNA using combination of various methods. DNA Extraction is a significant procedure in molecular biological methods and in forensic analysis. Several different extraction kits and protocols/methods are available. Proper extraction methods and procedures should be chosen to get the process optimized and also to save time.
Basic procedures for DNA extraction are as follows
- Break the cells open to expose DNA along with the cytoplasm.
- Removing membrane lipids by adding a detergent.
- Remove proteins by adding protease.
- The solution is then treated with saline to clump the debris (RNA, lipids, proteins) together.
- Centrifugation is performed to separate DNA from the debris.
- DNA purification is performed using ethanol.
- Isolate DNA and solubilize in Tris EDTA (TE) or ultrapure water. DNA is more stable in Tris EDTA.
Phenol Chloroform Extraction
Phenol Chloroform Extraction is a method used for extracting DNA from blood stains and liquid blood. Blood stains or liquid blood is placed in centrifuge tube. The stain extraction buffer (salts, detergents, buffer,10mm tris,100 mm Nacl, 39Mm Dithioreitol (DTT) ,10 Mm EDTA, 2% SDS (sodium dodecyl sulfate) is added. Salts and detergents helps solubilize cell components. DTT reduces and break apart disulfide linkages. Proteinase k is used to digest proteins. Phenol Chloroform isoamyl alcohol is added to form a milky emulsion and centrifuged to form bilayer. Aqueous layer contains the DNA, interphase contains the protein and proteins lipids can be seen in the organic layer. DNA can be precipitated using 100 % ethanol and then washed with 70 % ethanol solubilized in buffer
Cigarette butts, envelope flaps, saliva stains are treated as blood stains. In the case of hair, the hair must be rinsed in 100 % ethanol, water and rinsed in xylene. Bottom part (cm) of the hair is removed, treated with extraction buffer, proteinase k and should be treated as blood stain.
Bone powder (10 mg) should be incubated at 37c for 16 hours in 0.5ml EDTA for decalcification. Centrifuge and remove EDTA. Wash the powder with ultrapure water preferably 3 times. Extraction buffer and proteinase k was added along with the powder and incubated in shaking water bath at 56C for 8 hours. DNA is extracted using phenol chloroform isoamyl alcohol and should be purified using ethanol.