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Study Unit 1: Recombinant DNA Technology

• Professor: Rasheed Adeleke

• Subject: Microbiology - North-west University

• Focus: Recombinant DNA Technology and Industrial Microbiology

1. Overview of DNA Manipulation

1.1 Central Dogma

• Overview of DNA structure and replication

• Fundamental principles governing genetic information flow in cells

1.2 Obstacles in Genetic Engineering

• Historical challenges faced in genetic engineering

• Solutions developed to overcome these challenges

1.3 Steps in Microbial DNA Manipulation

• Procedures and techniques involved in manipulating microbial DNA

2. Steps in Microbial Gene Manipulation

Basic Techniques

• Construction of recombinant molecules known as genetic engineering

• Molecular cloning allows amplification of composite genes

3. Role of Vectors in Gene Manipulation

• Vectors are essential for DNA replication during cloning

• Common types of vectors include plasmids and bacteriophages

  • Suitable because they function independently without integrating into host genomes

4. Techniques Involved in Gene Manipulation

Agarose Gel Electrophoresis

• Process for purification of specific DNA fragments

• Separates DNA based on size (hundreds of base pairs to 20 kb)

Blotting Techniques

• Immobilization of nucleic acids on membranes for hybridization assays

• Common methods: dot blotting, slot blotting

Transformation Methods

• Essential for introducing plasmid DNA into E. coli cells

• Electroporation: alternative method to introduce DNA into cells that are not competent

5. Polymerase Chain Reaction (PCR)

Overview of PCR

• PCR allows exponential amplification of specific DNA sequences

• Utilizes DNA polymerases, primers, dNTPs, and specific thermal cycling

Steps of PCR Cycle

1. Denaturation: Heating to separate DNA strands

2. Annealing: Cooling allows primers to bind to target DNA

3. Extension: DNA polymerase synthesizes new DNA strands

Applications of PCR

• Amplification for sequencing, detection of specific DNA, and gene expression analysis

6. Types of PCR Techniques

Different PCR Types

• Conventional PCR: Basic amplification, easy to perform

• Multiplex PCR: Amplifies multiple targets simultaneously

• Real-Time PCR (QPCR): Quantifies DNA during the amplification process

• Nested PCR: Second-round amplification using specific primers to increase specificity

• RT-PCR: Amplifies RNA sequences into cDNA

• RT-QPCR: Combines RT-PCR with real-time quantification

7. Mechanisms and Components of PCR

• Reagents Required:

  • Buffer, template DNA, primers (forward and reverse), dNTPs, Taq polymerase

• Temperature cycling crucial for PCR success

Monitoring and Results

• PCR results in billions of identical DNA copies

• Importance of proper temperature and timing for successful amplification

8. Challenges and Solutions in PCR

• Considerations for choosing PCR types: reagent selection, thermal cycling parameters

• Potential for contamination and need for optimization of conditions

Summary of Learning Outcomes

After this study unit, you should be able to:

• Discuss various phases of DNA replication

• Illustrate the central dogma and its relevance in microbiology

• Develop a table or concept map of enzymatic and structural elements in gene manipulation

• Analyze obstacles in genetic engineering and basic techniques for resolution.