OCR A Level Biology:复习笔记2.3.4 DNA Purification

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

 

 

 

转载自savemyexams

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