Molecular techniques

• Molecular Techniques in Action


  Techniques used by molecular biologists to study DNA, RNA, and proteins
• Molecular biologists work at three levels: DNA, RNA, and Proteins
• DNA cloning


  Manipulation of DNA by cutting, joining, and moving DNA fragments into living systems

  
• DNA cloning


  1. Cut DNA at precise locations

  2. Use cloning vectors

  3. Join DNA fragments with DNA ligase

  4. Move DNA into living system

  5. Select and identify host cells containing recombinant DNA

  
• Restriction endonucleases (RE)


  Enzymes that recognise and cleave DNA at specific sequences (recognition sequences or restriction sites)
• Types of restriction enzymes

  • Type I
  • Type II
  • Type III
• Over 1000 REs have been identified
• Cohesive ends


  DNA fragments with 5' or 3' overhangs that can be joined by DNA ligase
• Blunt ends


  DNA fragments with no overhangs
• Compatible ends


  DNA fragments with complementary cohesive ends that can be joined by DNA ligase
• Palindromic DNA sequences


  DNA sequences that read the same forwards and backwards
• Restriction enzymes cut DNA with different frequencies depending on the length of the recognition sequence
• Frequent cutters


  Restriction enzymes with 4-8 bp recognition sites that cut DNA frequently
• Rare cutters


  Restriction enzymes with longer recognition sites that cut DNA less frequently
• Vectors


  Systems used to replicate and propagate DNA fragments
• Types of vectors and host systems

  • Plasmids in E. coli/S. cerevisiae
  • Bacteriophages in E. coli
  • Bacterial artificial chromosomes in E. coli
  • Yeast artificial chromosomes in S. cerevisiae
• Plasmids


  • Circular DNA molecules
  • Relatively small (2,000-6,000 bp)
  • Replicate separately from host chromosome
  • Contain RE recognition sequences
  • Contain antibiotic resistance genes
  • Contain origin of replication
  • Contain unique cloning sites
• DNA cloning using plasmids

  1. Restriction enzyme digestion

  2. Ligation

  3. Transformation

  4. Replication and selection
• Ligation


  The process of joining DNA fragments using DNA ligase, which repairs phosphodiester bonds and requires energy
• Transformation


  The process of introducing recombinant DNA into a host cell
• DNA library


  A collection of DNA fragments representing the entire genome or transcriptome of an organism
• Types of DNA libraries

  • Genomic DNA libraries
  • cDNA libraries
• cDNA libraries


  DNA libraries constructed from mRNA using reverse transcriptase, containing only the coding sequences of genes
• DNA cloning by PCR

  1. Heat to separate DNA strands

  2. Add primers

  3. Cool

  4. Add thermostable DNA polymerase

  5. Repeat cycles to exponentially amplify DNA
• PCR (Polymerase Chain Reaction)


  A technique that amplifies specific DNA sequences selectively
• Applications of PCR


  • Forensics
  • Diagnostics