Lecture 01, 2

• What are molecular methods used for in genetics?

  Identifying genes and their molecular function
• What is recombinant DNA technology?

  Technology that produces DNA by artificial means
• What is the purpose of introducing manipulated genetic material into a cell?

  To allow it to replicate and pass to progeny
• What are the key manipulations in recombinant DNA technology?

  • Cleavage of DNA at specific sites
  • DNA cloning
  • DNA ligation
  • Nucleic acid hybridisation
  • DNA synthesis
  • Rapid determination of nucleotide sequences
• What are endonucleases?

  Enzymes that cut inside a DNA molecule
• How do restriction endonucleases function?

  They recognise specific DNA sequences and cut them
• What type of sequences do Type II restriction enzymes recognize?

  Highly specific targets
• From where are restriction endonucleases mainly isolated?

  Bacteria
• What is a palindrome in the context of restriction endonucleases?

  Same sequence in anti-parallel orientation
• What is the typical length of a sequence recognized by restriction endonucleases?

  Usually 6bp long
• What do sticky ends created by restriction enzymes allow?

  Hybridisation of complementary DNA ends
• What is the role of ligases in DNA manipulation?

  Induce formation of phosphodiester bonds
• What are the types of vectors used in gene cloning?

  • Plasmids
  • Bacteriophages (viral vectors)
  • Artificial chromosomes
• How are vectors inserted into a host?

  Using electroporation or Ca2+ salts
• What is the purpose of including antibiotics in the medium for plating cells?

  To prevent growth of non-transformed cells
• What colour do blue colonies indicate in beta galactosidase culture?

  Enzyme activity without recombinant molecule
• What is the purpose of Polymerase Chain Reaction (PCR)?

  To amplify specific pieces of DNA in vitro
• What resources are needed for PCR?

  DNA template, primers, DNA polymerase, nucleotides
• What is Taq polymerase used for in PCR?

  To withstand high temperatures during amplification
• What happens to DNA during the heating phase of PCR?

  DNA strands separate at high temperatures
• How does agarose gel electrophoresis analyse PCR products?

  Separates DNA fragments based on size
• What does ethidium bromide do in gel electrophoresis?

  Illuminates DNA under UV light
• What are the applications of rDNA technology?

  1. Food & Agriculture
  2. Health & Diseases

  • Gene Therapy
  • Production of antibodies
  • Investigation of drug metabolism
  • Development of vaccines
  • Medically important compounds

  1. Environment/Industry
• What is Golden Rice and its significance?

  It contains beta-carotene to combat Vitamin A deficiency
• How is beta-carotene converted in the human body?

  Converted into vitamin A
• What is the genetic disorder associated with X-linked SCID?

  Defect in B and T cells of the immune system
• What is the drawback of gene therapy for ADA defective gene?

  Risk of leukemia
• What is the role of insulin in the body?

  Controls blood sugar levels
• What is Protropin used for?

  Treating growth hormone inadequacy
• What is the purpose of the recombinant HB vaccine?

  To prevent Hepatitis B infection
• What are the common applications of recombinant DNA technology in health?

  • Gene therapy
  • Production of antibodies
  • Development of vaccines
  • Production of recombinant hormones