8.4.1 Recombinant DNA technology

• What is 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

  1. Genetic code is universal

  2. Transcription and translation mechanisms are universal
• Describe how DNA fragments can be produced

  using restriction enzymes

  1. Restriction enzymes cut DNA at specific base ‘recognition

  sequences’ either side of the desired gene

  • Shape of recognition site complementary to active site

  2. Many cut in a staggered fashion forming ‘sticky ends’

  (single stranded overhang)
• Describe how DNA fragments can be produced from mRNA

  1. Isolate mRNA from a cell that readily synthesises the protein coded for by the desired gene

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

  
  

  3. DNA polymerase can form a second strand of DNA using cDNA as a template
• Suggest 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

  1. Mixture heated to 95 oC

  ● This separates DNA strands

  ● Breaking hydrogen bonds between bases

  2. Mixture cooled to 55 oC

  ● This allows primers to bind to DNA fragment

  template strand

  ● By forming hydrogen bonds between

  complementary bases

  3. Mixture heated to 72 oC

  ● Nucleotides align next to complementary

  exposed bases

  ● DNA polymerase joins adjacent DNA

  nucleotides, forming phosphodiester bonds

  This cycle is repeated - in every cycle, the amount of DNA doubles causing an exponential increase (2^n)
• Explain 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)
• Suggest 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

  1. Add promoter and terminator regions to DNA fragments

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

  restriction enzymes and ligases

  3. Transform host cells (eg. bacteria) by inserting these vectors

  4. Detect genetically modified (GM) / transformed cells / organisms by identifying

  those containing the marker gene (eg. that codes for a fluorescent protein)

  5. Culture these transformed host cells, allowing them to divide and form clones
• Explain why promoter and terminator regions are added to DNA fragments

  that are used to genetically modify organisms

  Promoter regions ● 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

  Terminator regions ● Ensure transcription stops at the end of a gene, by stopping RNA polymerase
• What are the 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

  1. 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

  2. DNA ligase joins DNA fragment to vector DNA

  ○ Forming phosphodiester bonds between adjacent nucleotides
• Describe how host cells are transformed using vectors

  ● 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
• Suggest how recombinant DNA technology can be useful in 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
• Suggest how recombinant DNA technology can be useful in 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)
• Suggest how recombinant DNA technology can be useful in 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
• Suggest some 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
• Suggest 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
• Suggest 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
• describe and explain how the PCR is used to amplify a DNA fragment

  • requires DNA fragment, DNA polymerase, DNA nucleotides and primers
  • heated to 95 degrees to break hydrogen bonds
  • cooled to 55 degrees to allow hydrogen bonds to form between primers and DNA
  • heated to 72 degrees to allow DNA polymerase to join nucleotides
• describe the growth of DNA as PCR repeats

  • increases exponentially as DNA doubles in each cycle
  • plateaus as no more nucleotides/primers
• suggest why it would be faster to create a gene using a gene machine than by using reverse transcriptase

  • more steps involved in reverse transcriptase
  • faster to use gene machine than all the enzyme catalysed reactions
• suggest one advantage of using fluorescent genes to identify bacteria that have taken up plasmids

  • quickly identify transformed bacteria using UV light