M6:S4 Manipulation genomes

• This section is all about techniques used to investigate and fiddle about with genes
• Techniques used to study genes and their function

  • The polymerase chain reaction (PCR)
  • Gel electrophoresis
  • Cutting out DNA fragments using restriction enzymes
• These techniques are also used in DNA profiling, DNA sequencing, genetic engineering and gene therapy
• Multiple Copies of a DNA Fragment can be Made Using PCR

  1. Set up reaction mixture containing DNA sample, free nucleotides, primers and DNA polymerase

  2. Heat to 95°C to break hydrogen bonds between DNA strands

  3. Cool to 50-65°C so primers can bind to strands

  4. Heat to 72°C so DNA polymerase can work

  5. DNA polymerase lines up free DNA nucleotides alongside each template strand

  6. Two new copies of the fragment of DNA are formed

  7. Cycle repeats, doubling the amount of DNA each time
• Polymerase chain reaction (PCR)

  • Can select a fragment of DNA and amplify it to produce millions of copies in just a few hours
  • DNA polymerase doesn't denature even at high temperatures, allowing many cycles of PCR
• Electrophoresis Separates DNA Fragments by Size

  1. Add gel tray to gel box/tank

  2. Add buffer solution to reservoirs

  3. Load DNA samples mixed with loading dye into wells

  4. Apply electrical current, causing DNA fragments to move through gel towards positive electrode

  5. Smaller fragments move faster and further through gel

  6. Stain gel to visualise DNA fragment bands
• Restriction enzymes

  Enzymes that recognise specific palindromic sequences (recognition sequences) and cut the DNA at these places
• Restriction Enzymes can be Used to Cut Out DNA Fragments

  1. Incubate DNA sample with specific restriction enzyme

  2. Enzyme cuts DNA fragment out via hydrolysis reaction

  3. Cut may leave sticky ends - small tails of unpaired bases
• Sticky ends can be used to bind the DNA fragment to another piece of DNA that has sticky ends with complementary sequences
• DNA profiling

  Analysing the number of times non-coding, repetitive DNA sequences are repeated at different loci in a person's genome
• The probability of two individuals having the same DNA profile is very low
• Forensic DNA profiling

  Comparing DNA samples collected from crime scenes to samples from suspects to link them to the crime
• Medical DNA profiling

  Analysing the risk of genetic disorders by identifying a broader, altered genetic pattern
• Genetic engineering


  The manipulation of an organism's DNA, often by inserting a gene from one organism into another
• How Genetic Engineering is Carried Out

  1. Obtain DNA fragment containing desired gene using restriction enzymes

  2. Insert DNA fragment into a vector (plasmid or bacteriophage) using restriction enzymes and DNA ligase

  3. Transfer vector into host organism, where it replicates and expresses the inserted gene
• Organisms with inserted genes from a different species are called transgenic organisms
• Transgenic organism


  An organism that has been genetically engineered to include a gene from a different species
• How Genetic Engineering is Carried Out

  1. The DNA Fragment Containing the Desired Gene is Obtained

  2. The DNA Fragment (with the Gene in) is Inserted into a Vector

  3. The Vector Transfers the Gene into the Bacteria
• Restriction enzymes


  Enzymes used to isolate a DNA fragment containing the desired gene
• Vector

  Something used to transfer DNA into a cell, e.g. a plasmid or bacteriophage
• DNA ligase

  Enzyme that joins the sugar-phosphate backbones of the vector DNA and DNA fragment
• Recombinant DNA


  The new combination of bases in the DNA (vector DNA + DNA fragment)
• Transformed


  Cells that take up the vectors containing the desired gene
• Genetic engineering can be used to benefit humans in loads of different ways
• Genetic engineering of plants

  • Can be used to create insect-resistance
  • Can lead to monoculture and decreased biodiversity
• Genetic engineering of animals

  • Can be used to produce drugs (pharming)
  • Raises concerns about animal welfare and the idea of animals as assets
• Genetic engineering of pathogens

  • Can be used to find treatments for disease
  • Raises concerns about potential for infection and biowarfare
• Technology transfer


  The sharing of knowledge, skills and technology between different institutions and scientists
• Patenting of genetically engineered products

  • Encourages competition and faster development
  • Can make products unaffordable for poorer farmers
• Gene therapy


  Altering alleles inside cells to cure genetic disorders
• Types of gene therapy

  • Somatic therapy
  • Germ line therapy
• Gene therapy


  • Potential to prolong lives and improve quality of life
  • Potential for short-lived effects and immune responses
• Germ line therapy in humans is currently illegal
• Somatic therapy


  Targets the epithelial cells lining the lungs
• Somatic therapy doesn't affect the individual's sex cells (sperm or eggs) though, so any offspring could inherit the disease
• Germ line therapy

  Involves altering the alleles in the sex cells, so every cell of any offspring produced from these cells will be affected by the gene therapy and they won't inherit the disease
• Positive Ethical Issues Surrounding Gene Therapy

  • It could prolong the lives of people with genetic disorders
  • It could give people with genetic disorders a better quality of life
  • Carriers of genetic disorders might be able to conceive a baby without that disorder or risk of cancer (only in germ line therapy)
  • It could decrease the number of people that suffer from genetic disorders (only in germ line therapy)
• Negative Ethical Issues Surrounding Gene Therapy


  • The technology could potentially be used in ways other than for medical treatment, such as for treating the cosmetic effects of ageing
  • There's the potential to do more harm than good by using the technology (e.g. risk of overexpression of genes)
  • There's concern that gene therapy is expensive - some people believe that health service resources could be better spent on other treatments that have passed clinical trials
• Potential Disadvantages of Gene Therapy

  • The effects of the treatment may be short-lived (only in somatic therapy)
  • The patient might have to undergo multiple treatments (only in somatic therapy)
  • It might be difficult to get the allele into specific body cells
  • The body could identify vectors as foreign bodies and start an immune response against them
  • An allele could be inserted into the wrong place in the DNA, possibly causing more problems, e.g. cancer
  • An inserted allele could get overexpressed, producing too much of the missing protein
• Germ line therapy - talking to a counsellor while in a queue at the doctors'