Biology (Year 12)
Image: Gel electrophoresis image, Image by Mckenzielower, Sourced Under a Creative Commons 4.0 License from Wiki Commons Gel electrophoresis is a biotechnological technique which is used to separate large, charged molecules (e.g. dyed fragments of DNA) according to size and charge. This allows us to visualise the DNA and identify it using a comparison with a standard. DNA molecules have a negative charge due to the sugar-phosphate backbone which is utilised in electrophoresis.
Image: Agarose gel electrophoresis image, Image by Microrao, Sourced Under a Creative Commons 4.0 License from Wiki Commons
A number of things are required to carry out electrophoresis.
Electric current (+ and - electrodes)
1. Setting up the apparatus: An agarose gel is used as the medium for electrophoresis, with 'wells' being made at the top of the gel using a comb. The gel is placed into the apparatus with a buffer solution for pH level regulation. The DNA we wish to visualise are cut using restriction enzymes into fragments of varying lengths and are dyed with a binding chemical such as fluorescent green dye, which fluoresces under UV light. (Note: Ethidium bromide used to be used, but is no longer used at it damages DNA and acts as a mutagen!) 2. Pipette samples: A 'DNA ladder' is pipetted into the well on the far left, and the other samples are placed in the other wells of the agarose gel using a micropipette. It is important to ensure that the samples are at the negative electrode end. Micropipette tips are to be discarded after every use. 3. Turning the electric current on: The negatively charged DNA molecules are repelled by the negative electrode and travel towards the positive end of the gel. Smaller DNA fragments move faster through the gel, and the larger DNA molecules move relatively slower. 4. Visualising the DNA and comparing it: The DNA fragments are then visualised by shining a UV light on the apparatus and photographing the results. The bands in each lane can be compared with the ladder to determine the length of the sample in terms of base-pair number. Gel electrophoresis has a number of applications.
Creating a DNA fingerprint to investigate crime scenes
Analysis of PCR results
Analysing genes associated with particular illnesses
Distinguishing species for taxonomic studies (DNA profiling)
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