• 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