2-3-4-dna-purification as

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