Techniques and Methods in CMB

• Paul Berg and others organized an international conference at the Asilomar Conference Center in Pacific Grove, California, to address the concerns regarding the use of recombinant DNA technology


  1975
• Asilomar Conference of 1975


  Brought together 140 molecular biologists, lawyers, government officials, and journalists with the support of the National Institutes of Health (NIH) and the National Academy of Sciences to discuss the potential risks associated with different types of experiments and methods to minimize these risks
• By the end of the Asilomar Conference meeting, an agreement was reached to lift the moratorium and allow research to continue within a set of defined restrictions
• Microscope


  An instrument that produces an enlarged image of an object
• Microscope


  • Has a light source, which may be external to the microscope or built into its base, to illuminate the specimen
  • Magnification refers to an optical instrument's ability to enlarge an image
  • Resolution is the ability of a piece of equipment to distinguish two objects and produce clearer images
  • Visibility, or contrast, is the difference in appearance between adjacent parts of an object or between an object and its background
• Bright-field microscope


  The cone of light that illuminates the specimen is seen as a bright background against which the image of the specimen must be contrasted
• Phase-contrast microscope


  • Converts differences in refractive index into differences in intensity (relative brightness and darkness), which are visible to the eye
  • Most useful for examining intracellular components of living cells at relatively high resolution
• Fluorescence microscope


  Allows viewers to observe the location of certain molecules called fluorophores, which absorb photons of a specific wavelength and release a portion of the energy in longer wavelengths (fluorescence)
• In the early 1960s, Osamu Shimomura discovered that a certain species of jellyfish (Aequorea victoria) owes its luminescent character to the presence of fluorescent proteins, such as aequorin and the green fluorescent protein (GFP), which he was able to purify and analyze
• Confocal microscope


  Produces an image of a thin plane within a much thicker specimen
• Transmission electron microscope (TEM)


  Forms images using electrons that are transmitted through a specimen
• Scanning electron microscope (SEM)


  Utilizes electrons that have bounced off the surface of the specimen
• Cell culture


  A laboratory method that enables the growth of eukaryotic or prokaryotic cells in physiological conditions
• Centrifugation


  A method of separating molecules having different densities by spinning them in solution around an axis (in a centrifuge rotor) at high speed
• Chromatography


  A biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis
• Types of chromatography


  • Ion-exchange or Affinity chromatography
  • Paper chromatography
  • Molecular-sieve chromatography
  • Agarose-gel chromatography
• Gel electrophoresis


  A laboratory method used to separate mixtures of DNA, RNA, or proteins according to molecular size
• Recombinant DNA technology


  Involves the use of enzymes and various laboratory techniques to manipulate and isolate DNA from different species or to create genes with new functions
• DNA cloning


  A molecular biology technique that makes many identical copies of a piece of DNA, such as a gene
• Polymerase Chain Reaction (PCR)


  A common laboratory technique used to make many (million or billion) copies of a particular region of DNA without the need for bacterial cells