DNA Purification
- Practical investigations can be conducted to purify (isolate) DNA via the process of precipitation
- Isolating DNA from cells is an essential starting point for a huge range of other investigations and so is a key research technique in the field of molecular biology
- A common method used to isolate DNA is known as the 'Marmur preparation'
- The method is derived from the work of Julius Marmur (1926-1996), an American molecular biologist who made significant contributions to DNA research
- The Marmur preparation involves three basic steps:
- Breaking (lysing) the cells and disrupting the nuclear membranes to release the DNA
- Using enzymes to denature and remove the proteins (histones) associated with the DNA
- Precipitating the DNA using an organic solvent (e.g. ethanol)
Example practical investigation: extracting DNA from onions
- Onions are good to use for this investigation as their cells contain a relatively large amount of DNA
- Fruits that also have relatively large amounts of DNA in their cells, such as strawberries, bananas and kiwis, can also be used
Equipment
- Plastic syringe (1 cm³)
- Plastic funnel
- 2 × beakers (250 cm³)
- 2 × Test tubes
- Stirrer (e.g. stirring rod or plastic spoon)
- Chopping board
- Knife (for chopping onion)
- Onion
- Washing-up liquid (10 cm³)
- Ice-cold ethanol (10 cm³)
- Protease enzyme (2-3 drops)
- Coffee filter paper (laboratory filter paper not suitable as the liquid takes too long to pass through)
- Water bath (60 °C)
- Ice-water bath
- Blender or liquidiser
Method
- Place the ethanol in a freezer 24 hours before starting the investigation
- The ethanol must be ice-cold, it is key to the success of the investigation
- Cut up the onion into small pieces (5 mm × 5 mm)
- Add the washing-up liquid to 90 cm³ of tap water in a beaker
- Add some of the onion pieces to the beaker
- Place the beaker in a water bath at 60 °C for 15 minutes
- The detergent (washing-up liquid) and the heat disrupt the phospholipid bilayer of the onion cell membranes and nuclear membranes, releasing the DNA
- The heat also denatures enzymes released from the cell that would otherwise begin to digest the DNA
- Cool the mixture in an ice-water bath for 5 minutes, stirring it continually
- Lowering the temperature prevents the DNA itself from breaking down, which would occur if the high temperature from the previous step was maintained
- Continual stirring ensures the whole mixture is cooled
- Pour the mixture into a blender and blend for 5 seconds
- Blending breaks down the cell walls and cell membranes of the onion cells even further, releasing more DNA
- The mixture is only blended for a very short time to ensure the DNA strands themselves are not broken apart
- Using the filter paper, filter the mixture into another beaker
- Filtering removes cell debris and membrane fragments
- The filtrate now contains the DNA and its associated proteins
- Pour 10 cm³ of the filtrate into a test tube and add 2-3 drops of protease enzyme, mixing well
- The protease denatures and removes the proteins, leaving just the DNA
- Carefully add the ice-cold ethanol to the test tube and wait 2-3 minutes
- Nucleic acids are insoluble in ice-cold ethanol and so the DNA forms a precipitate (white layer) at the top of the test tube mixture
Results
- The DNA in the resulting white precipitate can now be extracted and used for analysis or in further investigations
A method for isolating and extracting DNA from onion cells
