DNA profiling

DNA profiling

1. Tandem repeats are repetitive segments of DNA that do not code for proteins. The number of tandem repeats shows a family resemblance.

   • In DNA profiling methods short tandem repeats are compared. They are separated by electrophoresis.
     • STR are polymorphic - ask adams about this

2. Procedure

   • DNA obtained from individual e. mouth swab
     • DNA digested with restriction enzymes which cut the DNA at specific recognition sites into fragments of varying size
       • Fragments are separated by electrophoresis and stained. A banding pattern can be seen.
         • The DNA to which the individual is being compared to also undergoes the same process
           • Banding patterns can then be compared

3. Uses

   • Forensic science
   • Maternity and paternity disputes
   • Analysis of disease

4. PCR

   • Used to amplify DNA
     • It differs from DNA replication because it can only amplify short sequences, it requires a PRIMER, and a cycle of heating and cooling is needed
   • A cyclic reaction
   • The DNA polymerase is used from Thermophilus Aquaticus (it is called taq polymerase and can withstand high temperatures)
   • Process
     • 0. Sample is mixed with DNA nucleotides, primers, Mn2+, and tan DNA polymerase
       • 1. Heat to 94-96 to break H bonds (DNA is denatured into 2ssDNA)
         • 2. Cool to 68, primers can then anneal. small section of double strand is then on the ssDNA. This is so the taq polymerase can bind to it.
           • 3. Temperature raised to 72 (optimum for taq!), DNA remain as single strands, taq can then catalyse the addition of DNA nucleotides to the single strands.
             • It starts at the primer and moves in the 5' to 3' direction
             • The whole process repeats and is repeated for many cycles. The amount of DNA increases exponentially: 1 2 4 8 16 etc
   • Applications
     • Tissue typing prior to transplantation
     • detection of oncogenes
     • Detecting mutations (parents, fetal cells etc.)
     • Identifying viral infections in host cells genome (HIV)
     • Monitoring the spread of infectious disease
     • Research (extinct animals)

5. Electrophoresis

   • Process used to separate different sized fragments of DNA
     • Uses an agarose gel plate covered by a buffer solution
   • Process
     • 1. DNA samples cute at recognition sites by restriction enzymes. This is carried out at 35-40 and may take 1 hour
       • 2. Electrophoresis tank: agarose gel, combs at one end, buffer solution added. Removing the comb leaves wells.
         • 3. Loading dye is added to the digested DNA, this is then added to wells in the electrophoresis gel (pipette). Loading dye helps carry the DNA down into the well.
           • 4. Electrodes then put into place, connected to a 18v battery, this is left to run (higher voltage and high current can risk electric shock)
             • At the end, buffer solution is removed and a dye is added which adheres to the DNA and stains the fragments
   • Separating proteins
     • carried outings the presence of sodium dodecyl sulphate (SDS)
     • SDS is charged, it equalises the surface charge on the molecule and allows proteins to separate
     • This technique can be used to analyse haemoglobin proteins to diagnose conditions like sickle cell anemia, aplastic anaemia, thalassaemia, leukaemia
   • DNA PROBES
     • A DNA probe is a short single stranded length of DNA, complementary to a section of DNA being investigated
     • They can be labelled
       • radioactive marker (32 P) revealed by exposure to photographic film
       • fluorescent marker, emits colour on exposure to UV light
     • They usually anneal by complimentary base pairing
     • Uses
       • Locate a gene for use in genetic engineering
       • Genome comparison studies
       • to identify the presence/absence of an allele for a genetic disease
     • Micro arrays
       • Number of probes a fixed surface
       • Sample DNA anneals to complimentary fixed probes, this can reveal the presence of mutated alleles
       • Steps
         • 1. Sample DNA broken into fragments
           • 2. amplified using PCR
             • 3. Reference and test DNA samples are labelled with fluorescent markers
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