Module 5

• Expression cloning


  The creation of a perfect replica
• Expression cloning


  1. DNA coding for a protein of interest is cloned into a plasmid

  2. Introduction of DNA can be done by transformation, conjugation, or transduction

  3. Transfection - introduction of DNA into eukaryotic cells by physical or chemical means

  4. Plasmid may be integrated into the genome, or may remain independent of the genome

  
• Cloning vector


  • Small piece of DNA stably maintained in an organism
  • Where foreign DNA fragment is inserted
  • Genetically engineered

  
• Types of cloning vectors

  • Plasmids
  • Bacteriophage
  • Cosmids
  • Yeast artificial chromosome
• Restriction enzymes


  • Recognize specific DNA sequences and predictably cut them
  • Cuts palindromic DNA sequences
• Reporter genes


  Facilitate screening of successful clones
• Polymerase chain reaction (PCR)

  • Extremely versatile technique for copying DNA
  • Allows single DNA sequence to be copied millions of times

  
• Variations of PCR


  • RT-PCR for amplification of RNA
  • QPCR for quantitative measurement of DNA or RNA molecules

  
• Taq polymerase


  Thermostable DNA polymerase from Thermus aquaticus

  
• Polymerase chain reaction


  1. Denaturation (96°C) - Heat the reaction to separate DNA strands

  2. Annealing (55-65°C) - Cooling the reaction to enable binding of primers to complementary sequences on the ssDNA

  3. Extension (72°C) - Raising the reaction temperature so Taq polymerase extends primers and synthesize new DNA strands

  
• Gel electrophoresis


  Technique used to separate DNA fragments based on their size and charge

  
• DNA ladder


  Standard reference containing DNA fragments of known lengths
• Southern blot


  Probes presence of specific DNA sequence with DNA sample

  
• Northern blot


  Used to study the expression patterns of a specific type of RNA molecule

  
• Western blot


  Used to detect a specific protein in a blood or tissue sample

  
• Eastern blot


  Used to detect post-translational modification of proteins
• DNA array


  • A collection of spots attached to a solid support where each spot contains one or more single-stranded DNA oligonucleotide fragment
  • Each spot has a DNA fragment molecule complementary to a single DNA sequence
  • Useful for comparing gene expression of two different tissues