gene isolation

• Gene Isolation and Manipulation


  • In order to manipulate DNA or determine its sequence, the DNA needs to be massively amplified
  • In order to amplify and work with DNA, the DNA needs to be inserted into convenient DNA vectors (plasmids, phage)
  • Selection is the key! e.g., drug resistancy
  • Common sources of DNA: cDNA, genomic DNA, chemically synthesized DNA
• Restriction enzymes


  1. How to amplify an interesting gene

  2. How amplification works using DNA constructs that are propagated in bacteria
• Gel electrophoresis


  Can gel purify fragments prior to subcloning
• Cloning an insert into a vector

  1. DNA Palindrome GAATTC is EcoRI site

  2. Can use CIP (calf intestinal alkaline phosphatase) to remove 5' phosphate groups to prevent re-ligation of vector ends, OR use 2 different restriction enzymes
• Plasmid vectors


  • Can subclone fragments up to about 30 kb (but usually much smaller; also true for lambda)
  • lacZ encodes beta-galactosidase
  • Can express a His-tagged protein in bacteria (e.g., insulin!)
• Kary Mullis


  • Inventor of PCR
  • Obtained his PhD from the University of California, Berkeley
  • Came up with the idea while driving to his cabin retreat in Mendocino which he visited each weekend
  • Was working at Cetus Corporation, a biotech company
  • Was an avid surfer, and did a lot of LSD
  • Founded a business to sell pieces of jewelry containing amplified DNA of deceased famous people (e.g., Elvis)
  • Expressed disagreement with scientific evidence supporting climate change and ozone depletion or that HIV causes AIDS, and asserted his belief in astrology
  • Won the Nobel Prize in Chemistry in 1993
• More techniques in molecular biology

  1. cDNA synthesis by reverse transcription

  2. Producing cDNA molecules with sticky ends

  3. Producing PCR products with sticky ends
• Other vectors used to amplify DNA

  • Fosmid and BAC vectors carry large inserts
  • YACs allow even bigger inserts
• Modes of delivering recombinant DNA into bacterial cells

  Can make a "library" of genomic DNA from an organism
• Alz50


  An antibody directed against tau
• Production of human insulin by recombinant DNA technology

  Should be beta cell!
• Methods for detecting and quantifying DNA, RNA, and protein


  qPCR and qRT-PCR to get accurate quantifications!!
• Northern, Western, and Southern blots for insulin

  Could probe a new organism with a gene of interest to see if that organism contains a related gene....e.g., Hox genes!
• DNA sequencing: the ultimate goal for many genomics experiments. For example, sequencing is used to determine where human disease mutations are.

• DNA sequencing by the dideoxy method

  Cannot form phosphodiester bond with incoming dNTP!
• DNA sequencing by the dideoxy method

  Read from bottom!
• Results from automated sequencing


  Different fluorescent dyes (at end of each read) are read by the sequencer as the gel fragments are separated
• The Ti plasmid vector of Agrobacterium tumefaciens for making transgenic plants


  • Bacterium infects a plant cell, Ti plasmid is transferred and inserted
  • These techniques are used to generate pest-resistant crops!
• Producing a mouse containing the targeted gene knockout

  Tail prep and PCR to confirm presence of mutation!
• CRISPR/Cas9-mediated genome engineering


  Clustered, regularly interspaced short palindromic repeats-CRISPR-associated protein 9
• Manipulation of the mouse Ins1 gene by CRISPR-Cas9

  Enhanced transcription!