Genetics Ch.20- Recombinant DNA Technology Review

Recombinant DNA Technology

• Introduction to Recombinant DNA
  • Definition: Joining of DNA molecules from different biological sources not together in nature.
  • Clones: Recovered copies used to study the structure and orientation of DNA.
  • Purpose: Used to isolate, replicate, and analyze genes.

Key Tools in Recombinant DNA Technology

Restriction Enzymes

• Definition: DNA-cutting enzymes produced by bacteria to defend against bacteriophages.
• Function: Bind to DNA at specific sequences (restriction sites) and cleave it, creating restriction fragments.
• Cleavage Types:
  • Sticky Ends (Cohesive Ends): Fragments have overhangs.
  • Blunt Ends: Fragments have double-stranded ends.

DNA Ligase

• Function: Seals the phosphodiester backbone, joining restriction fragments covalently to make intact DNA molecules.

Vectors

• Definition: Carrier DNA molecules that replicate cloned DNA in host cells.
• Key Features:
  • Must replicate independently.
  • Include several restriction enzyme sites.
  • Carry selectable gene markers for distinguishing transformed host cells.

Plasmids

• Definition: Extrachromosomal double-stranded DNA molecules that replicate independently from chromosomal DNA in bacteria.

Transformation Process

• Plasmid introduction into bacteria can be achieved via:
  • Calcium ion heat shock.
  • Electroporation to facilitate DNA uptake.

Multiple Cloning Sites (MCS)

• Contains restriction sites for commonly used enzymes, allowing the cloning of different fragments.

Blue-White Screening

• Selectable Marker Genes: Provide antibiotic resistance.
• Mechanism:
  • Plasmid with the lacZ gene encodes galactosidase.
  • Agar plates with X-gal differentiate between recombinant (white) and nonrecombinant (blue) colonies.

DNA Libraries

Types of DNA Libraries

• Genomic Library: Contains at least one copy of all genome sequences.
• Complementary DNA (cDNA) Library: Contains cDNA made from mRNA, representing actively transcribed genes.

Construction of cDNA Libraries

• Steps:
  1. Isolation of mRNA.
  2. Synthesis of cDNA using reverse transcriptase.
  3. Cloning cDNA into vectors.

Polymerase Chain Reaction (PCR)

• Definition: A rapid method of DNA cloning that amplifies specific DNA sequences without host cells.
• Components Needed:
  • Two primers (complementary sequences).
  • DNA polymerase.
  • dNTPs.
• Three Steps of PCR:
  1. Denaturation.
  2. Primer annealing.
  3. Extension.

Molecular Techniques for Analyzing DNA and RNA

Restriction Maps

• Describe the number, order, and distances between restriction sites on DNA.
• Created using gel electrophoresis to separate DNA fragments by size.

Nucleic Acid Blotting Techniques

• Southern Blot: Identifies DNA sequences in cloned fragments.
• Northern Blot: Examines gene expression.
• Western Blot: Analyzes proteins.

Advanced Sequencing Techniques

Dideoxynucleotide Sequencing (Sanger Method)

• Most common DNA sequencing method, utilizing dideoxynucleotides to terminate DNA synthesis.

Automated DNA Sequencing

• Utilizes computer-automated systems for high-throughput sequencing, contributing to projects like the Human Genome Project.

Creating Knockout and Transgenic Organisms

Gene Targeting and Knockouts

• Gene targeting manipulates specific genes to study function.
• Knockout Mice: Mice with one or more genes disrupted to understand gene function.

Cre-Lox System

• Allows for conditional gene knockouts that can be controlled temporally and spatially.

Transgenic Animals

• Organisms that express or overexpress genes.
• Methods include homologous recombination into the host genome or injection into embryos.

CRISPR-Cas9 Technology

• A revolutionary gene editing technique that uses engineered nucleases for targeted modifications of DNA sequences, allowing precise changes to genes.