8.4.1 recombinant DNA technology

• recombinant DNA technology

  transfer of DNA fragments from one organism or species to another
• explain why transferred DNA can be translated within cells of recipient

  (transgenic) organisms

  genetic code is universal

  transcription and translation mechanisms are universal
• how DNA fragments can be produced using restriction enzymes

  restriction enzymes cut DNA at specific base ‘recognition sequences’ either side of the desired gene

  shape of recognition site complementary to active site

  many cut in a staggered fashion forming ‘sticky ends’ (single stranded overhang)
• how DNA fragments can be produced from mRNA

  isolate mRNA from a cell that readily synthesises the protein coded for by the desired gene

  mix mRNA with DNA nucleotides and reverse transcriptase → reverse transcriptase uses mRNA as a template to synthesise a single strand of complementary DNA (cDNA)

  DNA polymerase can form a second strand of DNA using cDNA as a template
• two advantages of obtaining genes from mRNA rather than directly from the DNA removed from cells

  much more mRNA in cells making the protein than DNA → easily extracted

  in mRNA, introns have been removed by splicing (in eukaryotes) whereas DNA contains introns

  so can be transcribed & translated by prokaryotes who can’t remove introns by splicing
• describe how fragments of DNA can be produced using a gene machine

  synthesises fragments of DNA quickly & accurately from scratch without need for a DNA template

  amino acid sequence of protein determined, allowing base sequence to be established

  these do not contain introns so can be transcribed & translated by prokaryotes
• name an in vitro and in vivo technique used to amplify DNA fragments

  in vitro (outside a living organism) -> polymerase chain reaction

  in vivo (inside a living organism) -> culturing transformed host cells eg bacteria
• explain how DNA fragments can be amplified by PCR

  mixture heated to 95 degrees -> this separates DNA strands, breaking hydrogen bonds between bases

  mixture cooled to 55 degrees -> this allows primers to bind to DNA fragment template strand, by forming hydrogen bonds between complementary bases

  mixture heated to 72 degrees -> nucleotides align next to complementary exposed bases, DNA polymerase joins adjacent DNA nucleotides, forming phosphodiester bonds

  cycle is repeated, in every cycle the amount of DNA doubles causing an exponential increase
• the role of primers in PCR

  primers are short, single stranded DNA fragments

  complementary to DNA base sequence at edges of region to be copied / start of desired gene

  allowing DNA polymerase to bind to start synthesis (can only add nucleotides onto pre-existing 3’ end)

  two different primers (forward and reverse) are required (as base sequences at ends are different)
• one reason why DNA replication eventually stops in PCR

  there are a limited number of primers and nucleotides which are eventually used up
• summarise the steps involved in amplifying DNA fragments in vivo

  add promoter and terminator regions to DNA fragments

  insert DNA fragments & marker genes into vectors (eg. plasmids) using

  restriction enzymes and ligases

  transform host cells (eg. bacteria) by inserting these vectors

  detect genetically modified (GM) / transformed cells / organisms by identifying those containing the marker gene (eg. that codes for a fluorescent protein)

  culture these transformed host cells, allowing them to divide and form clones
• why promoter regions are added to DNA fragments that are used to genetically modify organisms

  allow transcription to start by allowing RNA polymerase to bind to DNA

  can be selected to ensure gene expression happens only in specific cell types

  eg in gland cells of a mammal so the protein can be easily harvested
• why terminator regions are added to DNA fragments that are used to genetically modify organisms

  ensure transcription stops at the end of a gene, by stopping RNA polymerase
• role of vectors in recombinant DNA technology

  to transfer DNA into host cells / organisms

  eg plasmids or viruses (bacteriophage)
• explain the role of enzymes in inserting DNA fragments into vectors

  restriction endonucleases / enzymes cut vector DNA

  same enzyme used that cut the gene out so vector DNA & fragments

  have sticky ends that can join by complementary base pairing

  DNA ligase joins DNA fragment to vector DNA

  forming phosphodiester bonds between adjacent nucleotides
• describe how host cells are transformed using vector

  plasmids enter cells (eg following heat shock in a calcium ion solution)

  viruses inject their DNA into cells which is then integrated into host DNA
• explain why marker genes are inserted into vectors

  to allow detection of genetically modified / transgenic cells / organisms

  if marker gene codes for antibiotic resistance, cells that survive antibiotic exposure = transformed

  if marker gene codes for fluorescent proteins, cells that fluoresce under UV light = transformed

  as not all cells / organisms will take up the vector and be transformed
• how recombinant DNA technology can be useful -> medicine

  GM bacteria produce human proteins (eg insulin for type 1 diabetes) → more ethically acceptable than using animal proteins and less likely to cause allergic reactions

  GM animals / plants produce pharmaceuticals (‘pharming’) → cheaper

  gene therapy
• how recombinant DNA technology can be useful -> agriculture

  GM crops resistant to herbicides → only weeds killed when crop sprayed with herbicide

  GM crops resistant to insect attack → reduce use of insecticide

  GM crops with added nutritional value (eg golden rice has a precursor of vitamin A)

  GM animals with increased growth hormone production (eg salmon)
• how recombinant DNA technology can be useful -> industry

  GM bacteria produce enzymes used in industrial processes and food production
• describe gene therapy

  introduction of new DNA into cells, often containing healthy / functional alleles

  to overcome effect of faulty / non-functional alleles in people with genetic disorders eg cystic fibrosis
• issues associated with gene therapy

  effect is short lived as modified cells (eg T cells) have a limited lifespan → requires regular treatment

  immune response against genetically modified cells or viruses due to recognition of antigens

  long term effect not known - side effects eg could cause cancer

  DNA may be inserted into other genes disrupting them → interfering with gene expression
• why humanitarians might support recombinant DNA technology

  GM crops increase yields → increased global food production → reduced risk of famine / malnutrition

  gene therapy has potential to cure many genetic disorders

  ‘pharming’ makes medicines available to more people as medicines cheaper
• why environmentalists and anti-globalisation activists might oppose recombinant DNA technology

  recombinant DNA may be transferred to other plants → potential herbicide resistant ‘superweeds’

  potential effects on food webs eg affect wild insects → reduce biodiversity

  large biotech companies may control the technology and own patents