DNA Profiling

DNA Profiling

Short Tandem Repeats
1. Found within introns (intragenic regions) that are non-coding
  1. Inherited in the same way as any genes within the coding regions exons (expressed regions)
2. short DNA sequences repeated many times
3. Occur at the same locus on both chromosomes of a homologous pair
  1. the number of repeats on each of the homologous chromosomes can be different
  2. the number of repeats at a locus varies between individuals
    1. Large amount of variation in the number of repeats at each locus
      1. Therefore 2 individuals are highly unlikely to have the same combination of STR's
4. Obtaining the DNA
  1. Restriction Enzymes
    1. Endonucleases means they cut the DNA at sites within a strand of DNA
    2. Found naturally in bacteria where they cut up invading viral DNA
    3. Only cut DNA at specific base sequences, usually 4 or 6 base pairs long
    4. If restriction sites are either side of an STR sequence, that fragment of DNA will remain intact, but it will cut away the rest of the genome
      1. the repeated sequences remain intact
    5. The names of the restriction enzymes reflect the bacteria from which they originate
      1. Eg, EcoR1 comes from a strain of Ecoli
    6. They cut a DNA sample into fragments only where their specific restriction sequence occurs.
  2. Polymerase Chain Reaction
    1. the sample is placed in a reaction tube with DNA polymerase, DNA primers and nucleotides
      1. DNA primers are short DNA sequences complementary to the DNA adjacent to the STR
        They're marked with fluorescent tags
      2. the reaction tube goes into the PCR thermal cycler
        Heated to 95degreesC which seperates the double stranded DNA
        Cool to 55degreesC which optimises the binding of the primer to the target DNA sequence in the sample
        Heat to 70degreesC which is the optimum temperature for the heat stable DNA polymerase. The polymerases attach and nucleotides are added, extending the DNA from the primer
        After 2 cycles, copies that are just the STR sequence fragment are produced
        As the cycle continues, huge numbers of the targeted DNA fragments are produced
    2. Electrophoresis
      1. Visualising the fragments
        Gel is quite fragile and the DNA fragments are double stranded after electrophoresis if restriction enzymes have been used.
        Southern blotting is used to transfer the fragments to a more resilient nylon or nitrocellulose membrane.
        The membrane is placed directly onto the gel and a wad of dry absorbent paper placed on top.
        This acts as a wick to draw buffer solution up through the gel, carrying DNA fragments onto the membrane.
        During this process, the fragments maintain their positions relative to each other and are denatured into single strands, exposing the base sequences.
        The membrane is then incubated with an excess of a labelled DNA probe
        DNA probe: a short section of DNA with a base sequence complementary to the target DNA sequence that needs to be located
        After allowing time for the probe to bind to any complementary sequences (hybridise), any unbound probe is washed away
        Probes may be radioactive, labelled with radioactive phosphorus, or labelled with a fluorescent marker
        With a radioactive probe, the membrane is dried and placed next to X-ray film.
        The film blackens wherever the probe has bound with the DNA to form double-stranded fragments.
        If the probe is fluorescent, its position on the membrane can be visualised under UV light
        A single band occurs on the profile where a person's maternal and paternal chromosomes have the same number of repeats at a particular locus.
        2 bands occur on the profile if the 2 chromosomes have a different number of repeats at a locus
        There must be a reference profile for comparison
        The reference profile may come from a suspect in a murder investigation, a relative in the case of identifying a corpse, or from the parents when establishing paternity
        The DNA primers have a fluorescent tag attached to them, which allows the system to be automated
        As the DNA fragments with their attached fluorescent tags move through the gel they pass a laser, the dye in the tag fluoresces and the coloured light is detected
        This effectively gives a time that it has taken for the fragments to pass through the gel.
        Passing a separate set of fragments of known length through the gel allows the length of time for passage through the gel to be calibrated with fragment size
        Several STR loci can be analysed at once by using tags that fluoresce at different wavelengths giving different colours for each of the STR loci.
        A computer processes the information from the detector displaying the the results of the gel electrophoresis as a graph.
      2. DNA fragments are separated according to their size
      3. DNA placed on agarose or polyacrymalide, which provide a stable medium through which the fragments can move (porous)
        Gel submerged in a buffer solution, and connected to electrodes that produce a potential difference (voltage) across the gel
        The negatively charged DNA fragments migrate through the gel according to their overall charge and size
        In a given time, smaller fragments end up closer to the positive electrode
        A reference sample with fragments of known length may be added to the gel. This is known as a ladder or marker.
        The fragments are measured in base pairs
  3. can come from almost all biological tissue
    1. animal or plant
  4. Eg, cells in cheek swab, WBC's in blood smear, bone marrow in a skeleton, sperm left after a sexual assault
  5. Tissue sample broken down in buffer solution (includes salt and detergent) to disrupt the cell membranes
    1. the small suspended particles, including the DNA are separated from the rest of the cell debris by filtering or centrifuging
      1. Protease enzymes are incubated with the suspension to remove proteins
        Then, cold ethanol is added to precipitate out the DNA
        Several stages of washing the DNA in buffer solution follow
5. Inherited in the same way as allelles of a gene, with offspring receiving one repeated sequence randomly from each parent.
  1. Genetic profiling can be used for identification purposes, settling paternity disputes, identifying stolen animals and looking at variation and evolutionary relationships between organisms
    1. Is DNA profiling infallible?
      1. Widely used in legal proceedings
        Generally thought to produce a result that is almost unique to the individual and a near certain indication of guilt in criminal trials
        But because, the DNA profile analyses only a few repeated sequences, it is less likely to be completely unique
        This is a particular problem if individuals being tested are closely related.
        In court, a forensic scientist estimates the chance of seeing the same DNA profile in the general population; this is usually in excess of 1 billion
        (even for identical twins, due to the accumulation of mutations during a person's life)
The same principles of separating DNA fragments are used when screening for genetic conditions like cystic fibrosis, measuring genetic diversity or studying evolutionary relationships

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	Creado por Ella Middlemiss hace más de 7 años