Southern Blot

  • Created by: 08rmorris
  • Created on: 28-03-15 13:44
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  • Southern Blot
    • Used to detect a specific target sequence in samples of DNA
      • DNA samples separated in an electrophoresis gel is fragile, and the separated DNA fragments continue to diffuse through it blurring the bands
        • So, in a blot, the DNA is transferred to a stronger substrate, where it can be fixed
          • The target sequence is then detected using a DNA probe
            • A DNA probe is simply a short length of single-stranded DNA with a label attached
              • There are two common types of label used:
                • - A radioactively-labelled probe (synthesised using the isotope 32P) can be visualised using autoradiography
                • - A flourescently-labelled probe will emit visible light when illuminated with invisible ultraviolet light. Probes can be made to fluoresce with different colours
              • If a probe is added to a mixture of different pieces of single-stranded DNA it will anneal to any fragments of DNA containing the complementary sequence
                • forms regions of double-standed DNA (hybrid) the process is therefore called hybridisation
    • METHOD
      • 1. DNA is extracted from the source cells and amplified by PCR to make enough DNA for the hybridisation
        • The DNA samples are then digested by a restriction enzyme into many small fragments, and the fragments separated on an electrophoresis gel
        • 2. The gel is placed in an alkali solution, which breaks the H bonds between the DNA bases causing the strands to separate
          • 3. A thin sheet or nitrocellulose is placed on top of the gel and covered with a stack of paper towels, weighted down to make good contact.
            • The alkali solution is drawn up through the gel to the paper towels by capillary action, bringing the DNA with it.
              • The negatively charged DNA sticks to the positively charged nylon membrane
            • 4. After a few hours the DNA fragments are all transferred to the nylon sheet
              • The nylon sheet is separated from the del and treated with UV light to fix the DNA molecules
              • 5. The nylon sheet is placed in a plastic bag containing a solution of labelled probes and mixed thoroughly.
                • The probes will anneal by complementary base-pairing to DNA fragments in the nylon membrane that have a complementary sequence
                  • Forming hybrid DNA molecules stuck to the nylon sheet (but they can't be seen)
                • 6. The location of the hybrid DNA can be visualised by different methods, depending on the label used.
                  • If the probe were radioactive then they can be visualised by autoradiography, and the probes show up as band on photographic film
                    • If the probes were fluorescent then they can be visualised as bands of light when illuminated by ultraviolet light.
    • The southern blot has many uses
      • - to identify fragments containing a particular gene out of the thousands of restriction fragments formed from an entire genome
      • - to identify genes in one species that are similar to those of another species. Genes are remarkably similar in sequence form one species to another
        • this has aided the identification of human genes
      • - To identify the short DNA sequences used in DNA fingerprinting
      • - to screen for genetic diseases


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