DNA Profiling

DNA Profiling

1. Short Tandem Repeats

   • Found within introns (intragenic regions) that are non-coding
     • Inherited in the same way as any genes within the coding regions exons (expressed regions)
   • short DNA sequences repeated many times
   • Occur at the same locus on both chromosomes of a homologous pair
     • the number of repeats on each of the homologous chromosomes can be different
     • the number of repeats at a locus varies between individuals
       • Large amount of variation in the number of repeats at each locus
         • Therefore 2 individuals are highly unlikely to have the same combination of STR's
   • Obtaining the DNA
     • Restriction Enzymes
       • Endonucleases means they cut the DNA at sites within a strand of DNA
       • Found naturally in bacteria where they cut up invading viral DNA
       • Only cut DNA at specific base sequences, usually 4 or 6 base pairs long
       • 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
         • the repeated sequences remain intact
       • The names of the restriction enzymes reflect the bacteria from which they originate
         • Eg, EcoR1 comes from a strain of Ecoli
       • They cut a DNA sample into fragments only where their specific restriction sequence occurs.
     • Polymerase Chain Reaction
       • the sample is placed in a reaction tube with DNA polymerase, DNA primers and nucleotides
         • DNA primers are short DNA sequences complementary to the DNA adjacent to the STR
           • They're marked with fluorescent tags
         • 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
       • Electrophoresis
         • 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.
         • DNA fragments are separated according to their size
         • 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
     • can come from almost all biological tissue
       • animal or plant
     • Eg, cells in cheek swab, WBC's in blood smear, bone marrow in a skeleton, sperm left after a sexual assault
     • Tissue sample broken down in buffer solution (includes salt and detergent) to disrupt the cell membranes
       • the small suspended particles, including the DNA are separated from the rest of the cell debris by filtering or centrifuging
         • 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
   • Inherited in the same way as allelles of a gene, with offspring receiving one repeated sequence randomly from each parent.
     • Genetic profiling can be used for identification purposes, settling paternity disputes, identifying stolen animals and looking at variation and evolutionary relationships between organisms
       • Is DNA profiling infallible?
         • 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)

2. The same principles of separating DNA fragments are used when screening for genetic conditions like cystic fibrosis, measuring genetic diversity or studying evolutionary relationships