Gene Technologies

Gene Technologies

1. Techniques

   • Polymerase Chain Reaction
     • Artificial DNA replication
     • Relies on fact that DNA:
       • Is made of antiparallel backbone strands
       • Is made of strands with 5' end and 3' end
         • Grows only from 3' end
       • Bases pair up according to complementary base pairing rules
     • Not identical to natural DNA replication
       • Can only replicate short sequences
       • Addition of primers required for process to start
       • Cycle of heating & cooling used to separate & bind strands
     • Cyclic reaction
       • 1. DNA sample heated with supply of DNA nucleotides
         • 2. Heated to 95 degrees - break hydrogen bonds between strands
           • 3. Short lengths of primer added
             • 4. Temp reduced to 55 degrees, primers bind to form small sections of double stranded DNA
               • 5. DNA polymerase can bind to double-stranded sections
                 • 6. Temp raised to 72 degrees (optimum for DNA polymerase), extend strand by adding free nucleotides
                   • 7. When DNA polymerase reaches other end, new double-stranded DNA molecule generated
       • Can be repeated many times
   • Electrophoresis
     • Used to separate DNA fragments based on their size
     • 1. DNA sample treated with restriction enzymes to cut into fragments
       • 2. Placed into wells at -ve end of gel
         • 3. Gel immersed in tank of buffer solution & electric current passed through solution
           • 4. DNA -ve charged b/c of many phosphate groups so attracted to +ve electrode & diffuses through gel
             • 5. Shorter lengths move faster than longer lengths so move further
               • 6. Position of fragments shown with dye
   • DNA Probes
     • Short single-stranded piece of DNA that is complimentary to a section of the DNA being investigated
       • Labelled
         • Radioactive marker
         • Fluorescent marker
     • Can be added to sample of DNA fragments & will bind by complimentary base pairing - ANNEALING
     • Useful in locating specific sequences
       • To locate specific desired gene wanted for genetic engineering
       • To identify same gene on variety of different genomes, from separate species, when conducting genome comparison studies
       • To identify presence or absence of allele for particular genetic disease
   • Automated DNA sequencing
     • Same as PCR but within sequencing mixture some free nucleotides carry fluorescent marker
       • These nucleotides are modified so if added to chain, throws off DNA polymerase
     • Fragment lengths different sizes
     • Machine laser reads colour of marker (pass through same as electrophoresis)

2. Genetic Engineering

   • Recombinant DNA - Where DNA fragments from different organisms are combined into a single organism
   • Obtaining Gene to be Engineered
     • mRNA produced from transcription of gene can be obtained from cells where gene is expressed, used as template to make copy of gene
     • Synthesised using automated polynucleotide sequencer
     • DNA probe used to locate gene on DNA fragments & gene cut using restriction enzymes
       • Restriction enzymes cut through DNA at specific points
         • Cuts DNA only where a specific base sequence occurs (restriction site)
         • Enzyme catalyses hydrolysis of backbone
         • Gives staggered cut leaving some exposed bases - STICKY ENDS
         • Both DNA fragments need to have been cut with same restriction enzyme so sticky ends are complimentary
   • Placing Gene in a Vector
     • DNA ligase to seal into bacterial plasmid
     • Seal into virus genome or yeast chromosome
     • Often have to contain regulatory sequences of DNA, ensure inserted gene is transcribed in host cell
   • Getting Gene into Recipient Cell
     • Microinjection
     • Viral transfer
     • Liposomes
   • Bacteria
     • Gene placed in vector - bacterial plasmid
       • Complimentary sticky ends, join to form recombinant plasmid
       • Many cut plasmids simply reseal to reform original plasmid
     • Large quantity of plasmid mixed with bacterial cell
       • Addition of calcium salts & heat shock increases rate of take-up
     • Less than 0.25% of cells take up plasmid - those that do are 'transformed bacteria'
     • Conjugation
       • Copies of plasmid DNA passed between bacteria
       • Often carry genes associated with antibiotic resistance - speeds up spread of antibiotic resistance between bacterial populations
   • Case Studies
     • Human Insulin
       • Used to be extracted from pancreas of pigs - not identical to human insulin
       • Centrifugation methods to separate mRNA from right length of pancreatic tissue
         • Reverse transcriptase synthesises complimentary strand to mRNA
           • DNA polymerase & nucleotides, strand is built on using copied DNA as template
             • Produces copy of original gene called cDNA
               • Unpaired nucleotides added to each end to give sticky ends complimentary to plasmid
                 • Plasmids cut open & mixed with cDNA
                   • Some plasmids take up gene
                     • DNA ligase seals plasmids
                       • Plasmids mix with bacteria, some take up recombinant plasmids
       • Identifying transformed bacteria
         • Bacteria chosen to have resistance to two antibiotics
           • cDNA added in middle of one of these genes
           • Will no longer have resistance to one antibiotic if it has taken up cDNA
     • Golden Rice
       • Rich in beta-carotene (forms vitamin-A)
       • Usually in endosperm, gene for beta-carotene is switched off
       • Complex metabolic pathway but most enzymes already present in endosperm
       • Greenpeace argue against
         • Leads to reduction in biodiversity
         • Human food safety of engineered rice is unknown
         • GM rice could breed with wild-types and contaminate wild ride populations
   • Gene Therapy
     • Somatic Cell
       • Techniques to insert allele are difficult
       • Treatment is short-lived as cells die
       • Genetic manipulations restricted to patient only
     • Germline Cell
       • Into embryo
       • Delivery techniques more straightforward
       • All cells derived from gremlin cells will contain copy of functioning allele
       • Unethical to engineer embryo, not possible to know if allele introduced without unintentional changes
       • Genetic manipulations may be passed onto children

3. Comparing Genomes

   • Genes found in all or many living organisms gives clues of relative importance of such genes to life
   • Shows evolutionary relationships
   • Model effects of changes to DNA
     • E.g. mutation on gene obtained from yeast, also found in human genome (yeast is haploid so mutation always shown in phenotype)
   • Comparing genomes of pathogenic and similar non-pathogenic organisms can be used to identify genes that cause disease
   • DNA of individuals can be analysed to reveal alleles associated with increased risk of particular diseases