Dna isolation

STEPS IN DNA ISOLATION

1. DNA ISOLATION

2. INTRODUCTION DNA isolation  is a process of purification of DNA from sample using a combination of physical and chemical methods. The first isolation of DNA was done in 1869 by Friedrich Miescher . Methods used to isolate DNA are dependent on the source, age, and size of the sample. In general, they aim to separate DNA present in the nucleus of the cell from other cellular components. Isolation of DNA is needed for genetic analysis, which is used for scientific, medical, or forensic purposes.

3. Scientists use DNA in a number of applications, such as introduction of DNA into cells and animals or plants, or for diagnostic purposes, in  medicine  the latter application is the most common. On the other hand,  forensic science  needs to recover DNA for  identification  of individuals (for example rapists, petty thieves, accident, or war victims), paternity determination, and plant or animal identification. Presence of proteins, lipids, polysaccharides and some other organic or  inorganic compounds  in the DNA preparation can interfere with DNA analysis methods. They can also reduce the quality of DNA leading to its shorter storage life.

4. SOURCES Sources for DNA isolation are very diverse. Basically it can be isolated from any living or dead organism. Common sources for DNA isolation include whole  blood  , hair,  sperm  , bones, nails, tissues, blood stains,  saliva  , buccal (cheek) swabs, epithelial cells, urine, paper cards used for sample collection, bacteria, animal tissues, or plants. Stored samples can come from archived tissue samples, frozen blood or tissue, exhumed bones or tissues, and ancient human, animal, or plant samples.

5. PROCEDURE Isolation of DNA basically consists of four major steps. Preparation of a cell extract. Purification of DNA from cell extract. Concentration of DNA samples. Measurement of purity of DNA concentration .

6. 1. Preparation of a cell extract: To extract DNA from a tissue/cells of interest, the cells have to be separated and the cell membranes have to be disrupted. The "Extraction buffer" helps in carrying out these processes. Chemicals such as EDTA (Ethylene Diamine Tetra Acetate) which removes Mg 2+  ions that are essential for preserving the overall structure of the cell membrane, and SDS (Sodium Dodecyl Sulfate) which aids in disrupting the cell membranes by removing the lipids of the cell membranes are included in the extraction buffer.

7. Having lysed the cells, the final step in the preparation of a cell extract is removal of insoluble cell debris. Cell debris and partially digested organelles etc. can be pelleted by centrifugation leaving the cell extract as a reasonably clear supernatant.

8. 2. Purification of DNA from cell extract . In addition to DNA the cell extract will contain significant quantities of detergents, proteins, salts and reagents used during cell lysis step and RNA. A variety of procedures can be used to remove these contaminants, leaving the DNA in a pure form. The most commonly used procedures are: Ethanol precipitation. Phenol–chloroform extraction. Minicolumn purification.

9. 1. Ethanol precipitation Ethanol precipitation usually by ice-cold ethanol or  isopropanol . Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. Precipitation of DNA is improved by increasing of ionic strength, usually by adding sodium acetate.

10. 2. Phenol–chloroform extraction. Phenol–chloroform extraction in which phenol denatures proteins in the sample. After centrifugation of the sample, denaturated proteins stay in the organic phase while aqueous phase containing nucleic acid is mixed with the chloroform that removes phenol residues from solution. 3. Minicolumn purification. Minicolumn purification that relies on the fact that the nucleic acids may bind (adsorption) to the solid phase (silica or other) depending on the pH and the salt concentration of the buffer.

11. 3. Concentration of DNA samples The most frequently used method of concentration is ethanol precipitation. In the presence of salt and at a temperature of -20°C or less, absolute ethanol will efficiently precipitate polymeric nucleic acids. With a concentrated solution of DNA one can use a glass rod to pull out the adhering DNA strands while for dilute solutions precipitated DNA can be collected by centrifugation and redissolving in an appropriate volume of water.

12. 4. Measurement of purity of DNA concentration. DNA concentrations can be accurately measured by UV absorbance spectrometry. The amount of UV radiation absorbed by a solution of DNA is directly proportional to the amount of DNA sample. Usually absorbance is measured at 260 nm, at this wave length an absorbance of 1.0 corresponds to 50 µg of double-stranded DNA per ml.

13. With a pure sample of DNA the ratio of the absorbancies at 260 nm and 280 nm (A 260 /A 280 ) is 1:8. Ratios of less than 1:8 indicate that the preparation is contaminated, either with protein or with phenol.

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