Chapter 9 Biotechnology Part 2 Lecture Notes

Overview

• Continuation of biotechnology and genetic engineering from the previous lecture.
• Focus on biotechniques enabled by genetic manipulation and DNA analysis.
• Key topics: PCR, DNA sequencing, DNA fingerprinting, blotting techniques, transgenic organisms, and gene therapy.

Polymerase Chain Reaction (PCR)

• Purpose: To make copies of target DNA, especially when in short supply.
• Applications:
  • Forensic analysis: Amplifying DNA from crime scenes (e.g., blood or hair).
  • Evolutionary research: Amplifying ancient DNA (e.g., from amber-encased mosquitoes).
• Process:
  1. Mix PCR components: target DNA, Taq polymerase, primers, nucleotides.
  2. Use of a thermal cycler to change temperatures:
     • Heat to 95°C to separate DNA strands.
     • Cool for primer binding.
     • Heat to 72°C for polymerase activity.
  3. Repeat cycles (~40 times) to exponentially increase DNA copies.

DNA Sequencing

• Purpose: To determine the sequence of nucleotides in DNA.
• Notable Project: Human Genome Project, completed in 2003.
• Process:
  1. Prepare a PCR-like reaction with fluorescently labeled nucleotides.
  2. DNA fragments of varying lengths generated.
  3. Use electrophoresis and laser to detect sequence.

DNA Fingerprinting

• Purpose: To identify individuals based on unique DNA patterns.
• Applications:
  • Forensic analysis, paternity testing, microbial source tracing.
• Process:
  1. Extract DNA and cut with a restriction enzyme.
  2. Electrophoresis to separate DNA fragments by size.
  3. Compare banding patterns to deduce identity or relationships.
• Notes:
  • Uses non-coding DNA regions (e.g., VNTR) for variability.

Blotting Techniques

Southern Blot

• Purpose: Identify presence of specific DNA sequences (e.g., disease genes).
• Process:
  1. Cut DNA, separate with electrophoresis.
  2. Transfer to a membrane, add labeled probe.
  3. Detect presence with radioactivity or fluorescence.

Northern Blot

• Purpose: Detect specific RNA sequences.
• Process: Similar to Southern, but with RNA.

Western Blot

• Purpose: Identify presence of specific proteins.
• Process: Use antibodies as probes post-electrophoresis.

Transgenic Organisms

• Concept: Transfer genes between organisms using universal genetic code.
• Examples:
  • Tobacco plants with firefly genes.
  • Mice with glowing proteins.
  • Glow fish as consumer pets.
• Applications: Production of pharmaceuticals in animal milk.

Gene Therapy

• Objective: Cure genetic diseases by inserting healthy genes into patients.
• Example: SCID treatment via retroviral delivery of normal genes.
• Process:
  1. Insert healthy gene into virus.
  2. Introduce virus to patient's stem cells.
  3. Reintroduce modified cells to patient.
• Current Status: Ongoing clinical trials with promising results.

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These notes summarize the key points from the lecture, outlining various biotechnological techniques and their applications in science and medicine.