14 A Molecular Genetics and Recombinant DNA Technology Notes

Introduction to Molecular Genetics and Recombinant DNA Technology

• Focus on applying concepts from Biofree40 semester to recombinant DNA technology.
• Key Goals:
  • Identify molecular genetics tools derived from microorganisms.
  • Describe methods to create recombinant DNA molecules.
  • Explain methods for introducing DNA into prokaryotic cells.

Molecular Genetic Analytical Methods

• Utilize DNA replication processes for molecular analysis.
• Important Techniques:
  • PCR (Polymerase Chain Reaction): Amplifies specific DNA regions.
  • Process:
    • Denaturation: Heat DNA above 90°C to separate strands.
    • Primer Annealing: Primers bind to unique sequences.
    • Melting Temperature (Tm): Calculated as:
      T_m = 2 imes (A + T) + 4 imes (G + C)
    • Extension: DNA polymerase synthesizes a new DNA strand.
  • Applications:
    • Fragment length polymorphism analysis (VNTR).
    • Example problem: Determine biological relationships between siblings using band patterns.
  • Dideoxynucleotide Sequencing: A method where modified nucleotides halt DNA synthesis, allowing for sequence determination.
  • Use of dideoxynucleotide triphosphates (ddNTPs) that prevent further elongation.
  • Gel Electrophoresis: Used to visualize PCR results based on fragment size and charge.

Recombinant DNA Technology

• Genetic Engineering: Involves creating organisms to produce desired traits (e.g., bacteria producing human insulin).
• Methods of DNA Introduction:
  • Conjugation: Transfer of replicated DNA via pili between bacteria.
  • Transformation: Uptake of DNA from the environment by bacteria.
  • Transduction: DNA transfer mediated by bacteriophages.

Molecular Cloning Basics

• Key Steps:
  • Use of Restriction Enzymes to cut DNA at palindromic sequences.
  • Sticky Ends: Can anneal with complementary DNA; e.g., recognition by BamHI.
  • Blunt Ends: No overhangs, necessary for certain applications.
  • DNA Ligase: Joins cut DNA backbone via covalent bonds.
  • Generating Recombinant Plasmids: Incorporate foreign DNA via transformation into host organisms like E. Coli.

Plasmids as Cloning Vectors

• Essential features include:
  • Antibiotic resistance genes: e.g., ampicillin resistance.
  • Multiple Cloning Sites (MCS): Specific sequences for inserting foreign DNA.
  • Reporter Genes: Indicate successful transformation (e.g., LacZ for blue/white screening).
  • Blue/White Screening: White colonies indicate successful insertion of DNA; blue colonies indicate failure to insert.

Common Applications of Recombinant DNA Technology

• Practical applications in research and industry, particularly in producing proteins like insulin.
• Emphasis on gaining hands-on experience in laboratory settings for better understanding of genetic principles.