DNA technology

• What is a vector used for?

  Transporting DNA into a host cell
• How is a plasmid used in DNA technology?

  As a vector
• How can plasmids be useful?

  They nearly always contain antibiotic resistance genes
• How are sticky ends formed?

  Cut by the same restrictive endonuclease at the same recognition sites
• Which enzyme joins the sticky ends to the plasmid DNA?

  DNA ligase
• What is the new DNA molecule called?

  Recombinant DNA
• What happens during the transformation stage?

  Bacteria, plasmids and ice cold calcium chloride solution are mixed together and heat shocked so that the bacterial membrane is more permeable for plasmids to enter
• What percentage of bacteria cells take up recombinant plasmids?

  5%
• What are examples of gene markers used to identify recombinant plasmids?

  • Resistance to an antibiotic
  • Fluorescent protein
  • Enzyme whose action can be identified
• What is an example of an enzyme marker?

  Lactase
• How does lactase function as an enzyme marker?

  Lactase turns X-gal blue, if the gene has been interrupted by the DNA fragment, X-gal will remain colourless
• How are fluorescent markers used?

  Fluorescent gene is incorporated into the plasmid, if the bacterium has taken the gene up, it will not be fluorescent
• What is the main benefit of fluorescent markers?

  It is quicker- cells can be observed under microscopes rather than waiting for colonies to grow
• How does cloning work?

  As the bacteria is cloned, the plasmid contained the DNA fragment is also cloned
• What are the basic requirements for cloning bacteria?

  • Oxygen for aerobic respiration
  • Nitrogen for protein synthesis
  • Glucose/carbon for ATP synthesis
  • Temperature for enzyme activity