8.4.1 Recombinant DNA technology

• Recombinant DNA technology

  ● Transfer of DNA fragments from one organism or species, to another

  ● Transferred DNA can be translated within cells of recipient (transgenic) organism because:

  1. Genetic code is universal

  2. Transcription and translation mechanisms are universal
• Fragments of DNA can be produced by several methods, including:

  • conversion of mRNA to complementary DNA (cDNA), using reverse transcriptase

  1. mRNA isolated from a cell readily synthesised protein coded by desired gene
  2. Mix mRNA with DNA nucleotides and reverse transcriptase - to make a single strand of cDNA
  3. DNA polymerase forms second strand of DNA using cDNA as a template
• Advantages of using mRNA to make DNA fragment rather than restriction enzymes to cut gene from DNA

  ● More mRNA in cell than DNA → easily extracted

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

  ○ So can be transcribed/translated by a prokaryote who can’t remove introns by splicing
• Using restriction enzymes to cut a fragment containing the desired gene from DNA

  • Different restriction enzymes cut DNA at specific base ‘recognition sequences’
  • Shape of recognition site complementary to the active site
  • Many cut in a staggered fashion
  • Forming ‘sticky ends’ (single-stranded overhang)
• Method 3 - Creating the gene in a ‘gene machine’

  ● Synthesises fragments of DNA from scratch without need for a pre-existing DNA template

  ○ Amino acid sequence of protein determined allowing nucleotide sequence to established

  ● DNA fragments produced quickly / accurately

  ● No introns → can be transcribed/translated by a prokaryote who can’t remove introns
• The principles of the polymerase chain reaction (PCR) as an in vitro method to amplify DNA fragments

  Reaction mixture contains:

  • DNA fragment
  • DNA polymerase (taq polymerase)
  • (forward/reverse) primers
  • nucleotides
• PCR

  1. Strand separation - 95C

  ● Separates DNA strands

  ● By breaking hydrogen bonds between bases

  2. Primer binding - 55C

  ● Allows primers to bind to DNA fragment template strand

  ● By forming hydrogen bonds between complementary bases

  
  

  3. This cycle is REPEATED

  • This cycle is repeated. In every cycle, the amount of DNA doubles causing an exponential increase (2^n)
• 3. Synthesis of DNA - 72C

  ● Optimum temperature for DNA polymerase (taq polymerase)

  ● Nucleotides align next to complementary exposed bases

  ● DNA polymerase joins adjacent nucleotides forming phosphodiester bonds
• ● Cycle repeated 30-40 times to make many copies of DNA fragment

  ● In every cycle, amount of DNA doubles so number of molecules made = 2^n, where n = number of cycles

  • Causing an exponential increase

  ● Stops when run out of nucleotides / primers
• Roles of primers

  ● Primer = short, single-stranded DNA fragment

  ● Complementary to template DNA at edges of region to be copied / start of the desired gene

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

  ● Two different primers are required (forward and reverse)

  ○ Because DNA strands run in anti-parallel, but DNA polymerase can only run in one direction
• • In vivo method - Step 1: The addition of promoter and terminator regions to the fragments of DNA.
  • Promoter regions
  • DNA sequences that tell RNA polymerase when to start transcription to produce mRNA
  • Can be only activated in specific cell types → control which of body cells protein produced in
  • Terminator regions
  • Tell RNA polymerase when to stop
• Step 2: Restriction endonucleases and ligases used to insert DNA fragment into vectors

  • Restriction endonucleases: Same enzyme from producing DNA fragment - Vector DNA and DNA fragment cut using same restriction enzyme
  • Vector DNA and DNA fragment have complementary 'sticky ends' which forms complementary base pairing
• Sticky ends meaning: single stranded DNA ends with bases exposed
• Role of DNA ligases in in vivo method

  • DNA ligase joins DNA fragment to vector DNA
  • Forming phosphodiester bonds between adjacent nucleotides on sticky ends
• Transformation of host cells using these vectors. - adding plasmids to a host cell

  • addition of calcium ions (to neutralise the charge)
  • Heat shocked
• Step 4 - Marker genes used to detect genetically modified (GM) cells or organisms

  • Marker genes, inserted into vectors at same time as target gene, are added in order to identify which cells have the desired gene - not all cells / organisms will take up the vector and be transformed
• Gene markers can code for proteins which give:

  1. Resistance to an antibiotic (cells surviving exposure are transformed)
  2. Fluorescence (fluoresce under UV = transformed)
• 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

  Note - if body cells are altered, changes are not heritable. Gene therapy in gametes is currently illegal.

  Students should be able to relate recombinant DNA technology to gene therapy.
• 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
• Students should be able to

  • evaluate the ethical, financial & social issues associated with the use and ownership of recombinant DNA technology in agriculture, in industry & in medicine AND
  • balance the humanitarian aspects of recombinant DNA technology
  • with the opposition from environmentalists & anti-globalisation activists.
• Exam question

  The ADA gene is inserted into a virus. Give two advantages of using a virus in gene therapy

  • can enter cells / infect cells / inject DNA into cells
  • targets specific cells
  • replicates (in cells);
• Individuals who have been treated by this method of gene therapy do not pass the ADA gene to their children. Explain why [1 mark]

  • REPRODUCTIVE cells / Gametes do not have ADA allele / gene

  Link to Gametes, Meiosis (4.3 of spec)