21 - Recombinant DNA Technology

• What is a genome?

  An entire set of DNA, including all the genes in an organism
• Sequencing Projects:

  • Improvements in technology allow us to sequence the genomes in a variety of organisms
  • Gene sequencing methods only work on fragments of DNA, so you need to chop DNA into smaller pieces first & then put back into order
  • The Human Genome Project (2003) mapped the entire human genome for the first time
• What is the proteome?

  All the proteins made by an organism
• Sequencing the Genome can help identify their Proteins:

  • Some parts of genome code for specific proteins, some parts of DNA are non-coding
  • Simple organisms (i.e. bacteria) don't have much non-coding DNA
  • This means its relatively easy to determine the proteome from the genome
  • This is useful for medical research e.g. developing vaccines after identifying the protein antigens
• Why are some Organisms harder to translate the genome?

  • More complex organisms contain large sections of non-coding DNA
  • They also contain regulatory genes, which determine when genes that code for specific proteins should be switched on & off
  • This makes it more difficult to translate their genome into the proteome, as its hard for to find bits that code for proteins amongst the non-coding & regulatory DNA
• Updating Sequencing Methods:

  • Past methods were labour-intensive, expensive, and on a small scale
  • Now these techniques are often automated, cost-effective, and on a large scale
  • EXAMPLE: pyrosequencing is a recently developed technique that can sequence 400 million bases over 10 hours.
• What is Recombinant DNA Technology?

  1. Recombinant DNA technology involves transferring a fragment of DNA from one organism to another
  2. As genetic code is universal & transcription/translation mechanisms are pretty similar, DNA can be used to produce a protein in the cells of a recipient organism
• What are transgenic organisms?

  Organisms that contain transferred DNA
• Methods of obtaining DNA fragments (Reverse Transcriptase)

  1. mRNA molecules are numerous & complementary to the gene, so are easier to obtain
  2. mRNA molecules are used as templates to make lots of DNA
  3. The enzyme, reverse transcriptase, makes DNA from an RNA template (called complementary DNA - cDNA)
  4. mRNA is first isolated from the cell. Then its mixed with free DNA nucleotides & reverse transcriptase. The reverse transcriptase uses mRNA as a template to synthesise a new strand of cDNA

  
• Example of using Reverse Transcriptase to make cDNA:

  • Pancreatic cells produce the protein insulin
  • They have loads of mRNA molecules complementary to the insulin gene, but only 2 copies of the gene itself
  • Complementary mRNA to insulin is mixed with free DNA nucleotides & reverse transcriptase, making cDNA
• Methods of obtaining DNA fragments (using Restriction Endonuclease Enzymes)

  • Some sections of DNA have palindromic sequences of nucleotides i.e. antiparallel base pairs
  • Restriction endonucleases are enzymes that recognise specific palindromic sequences & cut DNA at the places
  • Different restriction endonucleases cut at different specific recognition sequences , because the shape of the sequence is complementary to the enzyme's active site

  
• Methods of obtaining DNA fragments (using Restriction Endonuclease Enzymes) (PART 2)

  • If recognition sequences are present at either side of the DNA fragment, you can use restriction endonuclease to separate it from the rest of DNA
  • DNA is incubated with the specific restriction endonuclease, which cuts out the DNA fragment via a hydrolysis reaction
  • Sometimes the cut leaves sticky ends (small tails of unpaired bases at each end of the fragment). Sticky ends can be used to bind the DNA fragment to another piece of DNA that has sticky ends with complementary sequences

  
• Methods of obtaining DNA fragments (Using a 'Gene Machine')

  1. Technology has been developed so fragments of DNA can be synthesised from scratch, without the need for a pre-existing DNA template
  2. A database contains the necessary information to produce the DNA fragment
  3. This means that the DNA sequence doesn't need to exist naturally - any sequence can be made
• How to obtain DNA fragments (using a 'Gene Machine')

  • The sequence that is required is designed
  • The first nucleotide in the sequence is fixed to some sort of support, e.g. a bead
  • Nucleotides are added step by step in the correct order, in a cycle of processes that includes adding protecting groups
  • Protecting groups prevents unwanted branching
  • Short sections of DNA called oligonucleotides (roughly 20 nucleotides long) are produced.
  • Once these are complete, protecting groups are removed & oligonucleotides are joined to make longer DNA fragments

  
• What is 'In vivo cloning'?

  Once you've isolated the DNA fragment, you need to amplify it (make lots of copies) so you have a sufficient quality to work with. In vivo cloning is where copies of the DNA fragment are made inside a living organism.
• Step 1 - The DNA fragment is inserted into a Vector:

  1. The DNA fragment is inserted into vector DNA (something used to transfer DNA into a cell). They can be plasmids or bacteriophages
  2. The vector nuclease is cut open using restriction endonuclease, so the sticky ends are complementary to the sticky ends of the DNA fragments
  3. The vector DNA & DNA fragments are mixed together with DNA ligase (called ligation)
  4. The new combo of bases in the DNA (vector DNA + DNA fragment) is called recombinant DNA

  
• Step 2 - The Vector Transfers the DNA Fragment into Host Cells:

  1. The Vector with the recombinant DNA is used to transfer the gene into host cells
  2. If a plasmid vector is used, host cells have to be persuaded to take it in
  3. In bacteriophage vector, it infects the host cell by injecting its DNA into it
  4. Host cells that takes up the vectors containing the gene of interest are said to be transformed
• How do Plasmid Vectors persuade host cells to take in the Plasmid Vector?

  • Host bacterial cells are placed into ice-cold calcium chloride to make the cell walls more permeable
  • The plasmids are added
  • The mixture is heat-shocked (heated to around 42C for 1-2 mins), which encourages the cell to take in the plasmid
• Step 3 - Identifying Transformed Host Cells

  • Only about 5% of host cells will take up the vector, so marker genes are used to identify the cell

  1. Marker genes can be inserted into vectors at the same time as the gene is cloned
  2. Host cells are grown on agar plates where there divide & replicate. This creates a colony of cloned cells
  3. The marker gene can either code for antibiotic resistance (agar plate contains antibiotic so only transformed cells survive) or are fluorescent (shows transformed cells under a UV light)
• How to get Transformed Host Cell to produce Proteins:

  1. Vector must contain a specific promoter & terminator region
  2. This may be present in the vector DNA or are added along with the fragment

  • Promoter Regions - DNA sequences that tell the enzyme RNA polymerase when to start producing mRNA
  • Terminator Region - tells it to stop
• What is the difference between In Vivo Cloning & In Vitro Cloning?

  In Vivo Cloning involves amplifying DNA inside a living organism whereas In Vitro Cloning outside a living organism
• How does In Vitro Cloning work: (PART 1)

  1. A reaction mixture is set up that contains the DNA sample, free nucleotides, primers & DNA
  2. The DNA mixture is heated to 95C to break the hydrogen bonds
  3. Mixture is cooled to 55C so the primers can bind to strand

  
• How does In Vitro Cloning work: (Part 2 )

  1. The reaction is heated to 72C, so DNA polymerase can work
  2. The DNA polymerase lines up free DNA nucleotides alongside each template strand & joins the nucleotides together. Specific base pairing means new complementary strands are formed.

  
• How does In Vitro Cloning work: (Part 3)

  1. Two new copies of the fragment of DNA are formed & one cycle of PCR is complete
  2. The cycle starts again, with the mixture being heated to 95C & this time all four strands (two original & two new) are used as templates
  3. Each PCR cycle double the amount of DNA

  
• What is genetic engineering?

  Transforming microorganisms, plants or animals with recombinant DNA to elicit a desired/favourable trait
• How is Recombinant DNA used in Microorganisms?

  In Vivo Cloning e.g. to produce insulin

  1. DNA fragment containing insulin gene is isolated
  2. DNA fragment is inserted into a plasmid vector
  3. Plasmid containing recombinant DNA is transferred into a bacterium
  4. Bacterium is identified & grown
  5. Insulin produced from the bacterium is extracted & purified

  
• How is Recombinant DNA used in Plants?

  • A gene that codes for a desirable protein is inserted into a plasmid
  • The plasmid is added to a bacterium & the bacterium is used as a vector to get the gene into plant cells
  • If the right promoter has been added with the gene, the transformed cells will be able to produce the desired protein
• How is Recombinant DNA used in Animals?

  • A gene that codes for a desirable protein can be inserted into an early animal embryo or into the egg cells of a female
  • If the gene is inserted into a very early embryo, all the body cells of the resulting transformed animal will end up containing the gene
  • If the gene is inserted into an egg cell, it means when the female reproduces, all the cells of her offspring will contain the gene
• How are proteins not produced in the wrong cells?

  • Promoter regions are only activated in specific cell types, meaning it can be controlled which cell the protein is produced in.
  • If the protein is produced only in certain cells, it can be harvested easier
  • Producing the protein in the wrong cell can damage the organism
• Recombinant DNA's benefits to Humans (Agriculture)

  • Crops can be transformed to give higher yield or be more nutritious, reducing the risk of famine or malnutrition
  • Crops can also be transformed to have pest resistance, leading to fewer pesticides being required
• Recombinant DNA's benefits to Humans (Industry)

  • Industrial processes often use enzymes.
  • These can be produced from transformed organisms, so they can produced in large quantities for less money
• Recombinant DNA's benefits to Humans (Medicine)

  • Many drugs & vaccines are produced by transformed organisms, using recombinant DNA technology
  • They can be made quickly, cheaply, and in large quantities using this method.
• Issues with using Recombinant DNA (Agriculture)

  • Farmers may create a monoculture, which makes the whole crop vulnerable to disease & reduces biodiversity
  • If transformed crops interbreed with wild plants, could produce 'superweeds', weeds resistant to herbicides
  • Organic farmers can have their crops contaminated by wind-blown seeds from modified crops
• Issues with using Recombinant DNA (Industry)

  • By making biotechnology companies more efficient, the companies get bigger & could force smaller companies out
  • Some people may not want to eat GM food
  • Consumer markets, like the EU, won't import GM foods
• Issues with using Recombinant DNA (Medicine)

  • Companies who own genetic engineering technologies may limit use of technologies that could be saving lives
  • Some people worry this technology could be used unethically i.e. making designer babies. Currently, this is illegal
• Humanitarian Benefits of Recombinant DNA technology:

  1. Agricultural crops can reduce the risk of famine & malnutrition
  2. Transformed crops can produce vaccines, making drugs more available to more people e.g. where refrigeration isn't available
  3. Medicines are more affordable
  4. Has the potential to be used on gene therapy to treat human diseases
• How does Gene Therapy work?

  1. Involves altering defective genes (mutated alleles) to treat genetic disorders and cancer
  2. Involves inserting a DNA fragment into a person's DNA

  • If the disease is caused by 2 mutated recessive alleles, you can add a dominant allele
  • If the disease is caused by a mutated dominant allele, you can 'silence' the dominant allele by sticking a bit of DNA in the middle of the allele so it doesn't work any more
• How do you insert the 'new' allele inside the cell?

  • The allele is inserted into cells using vectors just like recombinant DNA technology
  • Different vectors can be used e.g. altered viruses, plasmids, or liposomes
• What is Somatic Therapy?

  • It involves altering the alleles in body cells, especially in cells most affected by the disorder
  • EXAMPLE; cystic fibrosis damages the respiratory system, so somatic therapy for CF targets the epithelial cells lining the lungs
• What is Germ Line Therapy?

  • This involves altering the alleles in the sex cells
  • Therefore, every cell of the offspring produced from these cells will be affected by gene therapy & they won't suffer from the disease
  • GLT is currently illegal