Molecular Genetics - Practical Handbook Flashcards

Background Information

• Molecular Cloning: A set of methods used to create recombinant DNA molecules that can replicate in a host.

  • Historical Techniques: Early molecular cloning relied on PCR-restriction-ligation.
  • Modern Advancements: Ligation-free and recombination-based methods are frequently used now, especially for large-scale projects (e.g., cDNA libraries).

• Applications:

  • Protein Expression and Purification: Production of recombinant proteins (e.g., insulin, growth hormones).
  • Recombinant Vaccines and Genetically Modified Organisms: Such as knock-out mutants.

Cloning Vectors

• Integration of Foreign DNA: Starts with choosing a cloning vector, influenced by:

  1. Size of the foreign DNA
  2. Host organism (commonly E. coli)
  3. Final application (e.g., gene expression, or genetic modifications).

• Plasmid-based Vectors: Commonly used for small foreign DNA sequences.

  • Contains:
  1. Cloning Site (MCS): Where foreign DNA is inserted.
  2. Selectable Marker: Such as antibiotic resistance for positive selection.
  3. Propagative Elements: Functional origins of replication for E. coli growth.

• Additional Vector Elements Depending on Use:

  • Promoters, RBS (ribosome binding site) for expression.
  • Reporter genes, such as LacZα and GFP for fusions and visualization.

Polymerase Chain Reaction (PCR)

• Purpose: Amplifies target DNA for cloning.

  • For prokaryotic DNA, genomic DNA (gDNA) can be directly used as a template.
  • For eukaryotic DNA: mRNA is converted to cDNA using reverse transcriptase.

• PCR Master Mix: Typically includes:

  • DNA polymerase, dNTPs, and MgCl2 at a 2x concentration.
  • Adjusted with template and primers to alleviate contamination risk.

• PCR Cycle Stages:

  1. Denaturation (94-98°C, 10-30 sec)
  2. Annealing (48-68°C, 10-40 sec)
  3. Extension (72°C, 30sec - several minutes)

• Exponential Amplification: Each cycle doubles the target DNA, theoretically yielding up to 1.07 billion copies after 30 cycles.

Gel Electrophoresis

• Verification: Agarose gel electrophoresis checks PCR product size and purity.
  • Stained with SYBR® Safe or non-fluorescent stains for visualization.

Restriction Enzymes and DNA Ligation

• Restriction Endonucleases: Linearize cloning vectors and create compatible ends for DNA insertion.
• Ligation: T4 DNA ligase facilitates the covalent joining of vector and insert.
• Prevention of Self-ligation: Treat vector with Antarctic phosphatase to dephosphorylate ends prior to ligation.

Transformation of E. coli

• Methods of Transformation:

  1. Chemical Transformation (with CaCl2 and heat shock).
  2. Electroporation (electric field introduces DNA).

• Selection: Transformants grown on selective media where only cells with plasmids can survive.

Screening Transformants

• Colony PCR: Used to check for the presence of the foreign DNA insert in transformants.
• Vector and Insert Orientation: Important for downstream gene expression applications.

Expression of Recombinant Proteins

• Construct Transfer: Requires a suitable host for transcribing the recombinant gene.

  • Controlled by promoters (e.g., T7 system requires specific strains of E. coli).

• Detection Methods:

  • Electrophoresis: SDS-PAGE for separation followed by staining methods (e.g., Coomassie staining or Western blotting).

Experimental Design Overview

• Adrenal Secretory Protease (AsP): Study requires cloning the cDNA into a bacterial expression vector (e.g., pET32A).
• Fusion Protein: Enables detection and purification using His tags.

Cloning Steps**:

• Select appropriate restriction enzymes (NdeI, XhoI) for cloning into pET32A.
• Use primers designed for cloning that account for insertion sequencing and functional sites.
• Check transformant plasmids through colony PCR followed by restriction digestion verification.