8B genome projects and gene technologies

• Why can't the genome of a complex organism be easily translated into it's proteome

  • Non coding DNA is present
  • Regulatory genes  are present
• What is recombinant DNA?

  DNA of two different species / two types of organisms that has been combined 
• What are the 3 methods of producing DNA fragments?

  • conversion of mRNA to cDNA using reverse transcriptase
  • using restriction enzymes to cut a fragment containing the desired gene from DNA
  • creating the gene in a 'gene machine'
• Recall how reverse transcriptase can be used to isolate a gene. 

  • Cells that produce a large amount of mRNA for a required protein are identified
  • Cells centrifuged to break open + release mRNA
  • Free DNA nucleotides added to mRNA
  • cDNA strand formed using reverse transcriptase
  • Enzyme hydrolyses the mRNA (ribonuclease H)
  • mRNA and cDNA now separated, single strand of cDNA
  • DNA polymerase then catalyses formation of complementaty strand to the cDNA; producing a double helix of DNA

   
• What are endonucleases?

  Enzymes produced by bacteria to cut up viral DNA as a defensive measure 
• Recall how restriction endonucleases can be used to produce DNA fragments.

  • Cut DNA at specific sites, about 4-8 base pairs long, calledrecognition sites
  • Recognition sites are usually palindromic
  • RE makes staggered cuts will leave exposed bases on either end (sticky ends)
• Recall how we use a 'gene machine' to produce DNA fragments.

  • Sequence of bases determined
  • Triplets worked out + fed into computer
  • Computer designs oligonucleotides, which are assembled into the desired gene
• Once we have synthesised a gene, how is it amplified in vitro?

  PCR (polymerase chain reaction)
• What are the 2 ways in which gene cloning can be carried out?

  • in vivo - genes inserted into organism that produces copies during normal DNA replication
  • in vitro - genes duplicated in a machine using enzymes and rapid temperature changes (e.g PCR)

   
• Sticky ends are used in genetic engineering. Explain how

  • Joining two pieces of DNA;
  • By complementary binding/complementary base-pairing;
• Why are the same restriction endonucleases used to cut the DNA and the vector?

  ensures the sticky ends of the gene + plasmid are complementary to each other 
• What is the promoter region?

  • sequence within DNA where RNA polymerase binds
  • required in order to begin transcription
• What is the terminator region?

  • sequence within DNA that causes RNA polymerase to be released and ends transcription
• What is a vector?

  • Carrier of DNA
  • into cell / host / other organism
• Describe how PCR is carried out.

  •  DNA heated to 90 to 95°C;
  •  strands separate;
  •  cooled / to temperature below 70°C 
  •  primers bind;
  •  nucleotides attach;
  •  by complementary base pairing;
  •  temperature 70 - 75°C;
  •  DNA polymerase joins nucleotides together;
  •  cycle repeated;
• What is the role of primers in PCR?

  enables replication to start by keeping strands separate
• Recall the 5 stages involved in genetic engineering

  Isolation: genes isolated

  Insertion: insertion of gene into a vector

  Transformation: transfer of DNA into a host

  Identification: analysing to see which host cells have taken up gene using gene markers

  Growth/cloning: population of host cells rapidly increased
• Recall how a gene is inserted into a plasmid vector.

  • Sticky ends are complementary (since same REnds are used)
  • Promoter and terminator regions are added
  • DNA ligase forms 2 phosphoidester bonds
• What is the potential problem with the gene of interest/plasmid having the same sticky ends?

  Mismatch - self coiling of plasmid / gene inserted has combined to form new piece of DNA (circularised DNA)

   
• What are the two methods of transformation? (insertion of vector into bacteria)

  • Calcium ions added + heat shock
  • Electroporation
• Recall how calcium ions + heat shock are used to insert a vector into a bacteria.

  • Calcium ions added
  • Cells heat shocked
  • Increases permeability of membrane
• Recall how electroporation is used to insert a vector.

  • DNA negatively charged
  • Small electrical field increases permability
  • Causes movement of vector into host
• Why do only a small % of host cells have the required vector?

  • Not all plasmids incorporate donor DNA during insertion 
  • Not all cells incorporate vectors during transformation
• What are the 3 methods of identifying which host cells have taken up the vector?

  • GFP tagging
  • Enzyme marker
  • Antibiotic resistance: insertion and disruption
• How is GFP tagging used to identify desired host cells?

  • DNA sequence for GFP inserted into plasmid/donor DNA
  • Will light up green

   
• How are enzyme markers used to identify transgenic host cells?

  • Sequence for enzyme inserted / disrupted in either plasmid or donor DNA
  • E.g lactase is disrupted by insertion of DNA
  • Lactase turns colourless substrate blue hence transgenic bacteria will be clear colonies
• What is combination gene marking?

  • GFP / enzyme marker added to both plasmid DNA and Donor DNA
  • Allows us to identify hybrid plasmid
• What is replica plating?

  • Technique used to identify transgenic host cell
  • Plasmid used contains two antibiotic resistance genes
  • One of these is used as the site of insertion and is disrupted
  • Add both antibiotics to samples of bacteria
  • Bacteria w/ recombinant plasmid will be resistant to one, but not the other
• What is genetic fingerprinting?

  A technique which uses the individuality of DNA molecules to distinguish between organisms or show the relationship between them. Consists of PCR + Gel electrophoresis. 

   
• What are VNTRs?

  • Regions found in the non-coding part of DNA
  • Contain variable numbersof repeated DNA sequences and vary between diff people
  • VNTR may be referred to as 'satellite' or 'mini-satellite' DNA
• How are differences between VNTRs detected?

  • DNA sequences amplified via PCR
  • Sample separated by size via gel electrophoresis
• Recall the process of genetic fingerprinting

  • DNA extracted from sample
  • DNA hydrolysed into segments using restriction endonucleases
  • Must leave minisatellites / required core sequences intact
  • DNA fragments separated using electrophoresis
  • detail of process e.g. mixture put into wells on gel and electric current passed through
  • immerse gel in alkaline solutionhence two strands of DNA separatedradioactive marker / probe added  / complementary to VNTRs autoradiography;
  • Southern blotting / cover with nylon / absorbent paper (to absorb DNA)
  • (areas with probe) identified using X-ray film / 
• The probability of two individuals having the same VNTRs is...

  very low unless they're identical twins
• Recall 4 uses of genetic fingerprinting

  1. Identifying genetic relationships in paternity test
  2. Matching DNA from a crime scene to suspects
  3. Diagnosing diseases like Huntington's (based on number of AGC repeats on chromosome 4)
  4. Prevent undesirable inbreeding in farms or zoos. 
• What is a DNA probe?

  short, single-stranded length of DNA that is complementary to a known base sequence
• What are the two common forms of DNA probes?

  • Radioactively labelled probes - made up of nucleotides with isotope P32 . Identified via X-ray film.
  • Fluorescent labelled probes - emit light under certain conditions
• How are DNA probes used to locate specific alleles of genes?

  1. Sequence for mutated allele identified
  2. Probe made with complementary bases
  3. Probe is labelled (fluorescent or radioactive) then replicated
  4. DNA sample obtained
  5. DNA made single stranded by heating
  6. Probe added; joins by complementary base pairings. 
  7. Wash to remove unattached probes
  8. Can now identify using X-ray imaging or UV light
• What are the 4 main uses of DNA probes?

  • Genetic screening for diseases
  • Personalised medicine; choosing most effective treatments
  • Genetic counselling
  • Genetic fingerprinting
• What is gene therapy?

  treating genetic disease by providing the sufferer with a corrected copy of their defective gene
• What are the two methods of gene therapy currently used?

  1. Somatic cell therapy (body cells)

  2. Germ line therapy (gametes)