manipulating genomes

• what is a genome?

  all genes possessed by individual or organism

  -              All DNA in nucleus, mitochondria
• what is an exon?

  Express, code for proteins, 2% of genome
• what is an intron?

  Non – coding DNA, 98% of genome

  -              Removed form mRNA before leaving nucleus
• define a gene?

  section of DNA codes for protein resulting in a characteristics
• define satellite DNA, give examples?

  highly repetitive DNA with short sequences repeated large number of times

  -              Different people, different satellite patterns as different number of repeats

  -              More closely related, more similar pattern or satellite regions as size/ length of satellite DNA is inherited from parents

  -              Less related, more variation
• types of satellite DNA?

  1. Minisatellites:

   Variable Number Tandem Repeated (VNTR)

  -              20-50 base pairs long

  -              Repeated 50 – several hundred times

   

  2. Microsatellites: Shot Tandem Repeated (STR)

  -              2 – 4 base pairs long

  -              Repeated 5 – 15 times
• what is DNA profiling?


  image of patterns in non – coding DNA of individual

  -              DNA fingerprint (unique)
• steps of DNA profiling?

  1.        Extract DNA from sample (hair) – PCR used to amplify if too small

  2.        Digest sample – DNA cut into small fragments by enzyme: restriction endonuclease

  3.        Gel Electrophoresis: DNA fragments separated

  4. DNA separated to single strands by alkaline solution - H bonds broken

  5. Southern Blotting: DNA fragments transferred to nylon membrane

  6. Hybridisation: DNA probes added to label fragments (radioactive/ fluorescent)

  7. Development: radioactive tags placed into X – Ray film, developed, florescent tags: placed under UV light
• what is Gel Electrophoresis?

  -              DNA fragments loaded/ placed into well at top of agar gel

  -              Covered in buffer solution

  -              Electrical current applied DNA repelled from negative end, attracted to positive end from negative phosphate backbone of DNA

  -              Agar acts as ‘mesh’: resists movement of DNA fragments through

  -              DNA moves to positive electrode at different rates: smaller fragments quicker
• what DNA used in DNA profiling?


  Satellite DNA
• what enzyme is used in DNA profiling


  restriction endonuclease 
• what is PCR and stands for?

  Polymerase Chain Reaction

  -              Artificial DNA replication

  -              DNA fragments amplified

  -              Thermal cycler (PCR machine): controls, changes temperature at programmed timing to trigger different steps in PCR
• steps in PCR?


  1. preparation: denaturation, annealing, synthesis
  2. cycle replaced
• descibe preparation phase?

  1st step in PCR:

  4 DNA bases, small primer DNA sequences, DNA polymerase from Taq Polymerase (doesn’t denature at the temps) from thermophile bacteria into a vile, into Thermal cycler:
• 1st step in preparation of PCR?

          Denaturation: 95oC, DNA denatures: 2 strands separate, nitrogenous bases separate from complementary pair: H bond breaks, gain kinetic energy, vibrate more
• 2nd step in preparation of PCR?

  Annealing: Thermal cycle temp drops: 55oC, primers (short DNA sequences) bind to start of target DNA needing to be amplified as have complementary sequence
• 3rd step in preparation of PCR?

          Synthesis: temp increased to 72oC, free nucleotides pair up to exposed bases (complementary base paring), Taq polymerase form phosphodiester bonds on DNA backbone
• 2nd step of PCR?

          Cycle repeated: cyclic reaction
• what is PCR used for?

  -              Tissue typing

  -              Detection of oncogenes

  -              Detecting mutations

  -              Identifying viral infections

  -              Monitoring spread of infectious disease

  -              Forensic sciences

  -              Research
• advantages of PCR?


  very rapid, not require living cells