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