In Vivo Gene Cloning

Jul 8, 2024

Lecture: In Vivo Gene Cloning

Introduction

  • In Vivo Gene Cloning: Using bacteria to copy DNA
    • Gene cloning: Copying a gene
    • In vivo: “In life”, using living organisms (bacteria)

Process Overview

  1. Insert DNA into Bacterial Genome
  2. Incorporate DNA into Plasmid
  3. Bacteria Copies Plasmid
  4. Extract and Isolate Gene

Detailed Steps

DNA Fragmentation

  • Identify the gene to be copied within a larger DNA fragment
  • Use enzymes to cut the DNA
    • Polymerase Chain Reaction comparison: Starts with a fragment already
    • In Vivo Cloning: Position within a larger DNA is important

Bacterial Genome and Plasmids

  • Bacteria

    • Prokaryotic (no nuclei)
    • Main genome: One large DNA molecule
    • Plasmids: Small rings of DNA, multiple copies within bacteria

  • Plasmids

    • Continuously copied by bacteria
    • Attractive for molecular biology due to consistent replication
    • Not inserted into the main genome but into plasmids

Cutting and Inserting the Gene

  • Plasmid must be cut to insert gene
  • Nature’s Role: Restriction Enzymes
    • Bacteria produce restriction enzymes to defend against viruses
    • Example: Bacteriophage virus injects DNA into bacteria
      • Bacterial restriction enzymes cut viral DNA

Restriction Enzymes

  • Specificity: Recognize specific sequences and cut there
    • Example: EcoR1 found in E. coli
    • Recognizes and cuts G-A-A-T-T-C sequence, creating sticky ends
    • Sticky ends allow specificity for rejoining DNA
  • Sticky Ends: Cuts in a staggered way, creating overlaps
    • Higher specificity compared to blunt ends

Base Pair Palindrome

  • Base Pair Palindromic Sequence: Recognized by restriction enzymes
    • Reads the same forwards and backwards with complementary bases
    • Example Sequence: G-A-A-T-T-C
  • Anti-parallel DNA Structure: Strands run in opposite directions
    • Facilitates enzyme recognition from either DNA side

Enzyme Types

  • Restriction Endonucleases
    • Named for their function: Enzymes (ending in -ase)
    • Endonuclease: Cuts DNA mid-strand (not at ends)
    • Ectonuclease: Cuts nucleotides off from the ends

Conclusion

  • Recap of in vivo gene cloning process using restriction endonucleases
  • Preview: Continued discussion in next video