Biotechnology Principles and Processes

Principles of Biotechnology

Definition

Biotechnology involves the use of living organisms or their components to develop products and processes for specific applications.

Key Concepts

• Genetic Engineering: Manipulation of an organism’s DNA to alter its characteristics.
• Recombinant DNA Technology: Combining DNA from different sources to create new genetic combinations.

Tools of Recombinant DNA Technology

1. Restriction Enzymes

• Definition: Enzymes that cut DNA at specific sequences, known as recognition sites.
• Function: They create DNA fragments that can be used for cloning and analysis.
• Types:
  • Type I: Cut DNA at random sites and require ATP for activity.
  • Type II: Cut DNA at specific sites, commonly used in cloning.
    • Example: HindIII is a Type II restriction enzyme that recognizes the palindromic sequence 5’-AAGCTT-3’ and cuts between the A and the G, producing sticky ends that can facilitate the cloning process.

2. Palindromic Sequences

• Definition: A palindromic sequence in DNA is a sequence of base pairs that reads the same in the 5’ to 3’ direction on one strand and the 3’ to 5’ direction on the complementary strand.
• Importance: Many restriction enzymes recognize palindromic sequences, allowing them to bind and cut DNA at specific sites. For example, the recognition site for HindIII (5’-AAGCTT-3’) is palindromic because the reverse complement (3’-TTCGA-5’) reads the same in the opposite direction.

3. Endonucleases and Exonucleases

• Endonucleases: These enzymes cut the phosphodiester bond within a DNA strand. They can create either blunt or sticky ends, depending on the enzyme used.

  • Example: HindIII is an endonuclease that produces sticky ends.

• Exonucleases: These enzymes remove nucleotides from the ends of a DNA molecule. They can be used to trim DNA fragments or to degrade DNA from the ends.

  • Example: Exonuclease I selectively degrades single-stranded DNA from the 3’ end.

4. Separation and Isolation of DNA Fragments

• Techniques:
  • Gel Electrophoresis: A method to separate DNA fragments based on size. DNA is loaded into a gel and an electric current is applied, causing the fragments to migrate.
  • Purification: Isolating specific DNA fragments from the gel for further use.

5. Features Required for Cloning into a Vector

• Origin of Replication: Allows the vector to replicate within the host.
• Selectable Markers: Genes that confer resistance to antibiotics or other markers to identify successful transformants.
• Multiple Cloning Site (MCS): A region containing several restriction sites for inserting foreign DNA.

6. Competent Host

• Definition: A host cell that can take up foreign DNA through transformation.
• Methods to Create Competent Cells:
  • Chemical treatment (e.g., calcium chloride).
  • Electroporation (using an electric field to increase permeability).

Processes of Recombinant DNA Technology

1. Isolation of the Genetic Material (DNA)

• Methods:
  • Cell lysis to break open cells and release DNA.
  • Use of detergents and enzymes to digest cellular components.

2. Cutting of DNA at Specific Locations

• Using Restriction Enzymes: DNA is cut at specific sites to create fragments that can be manipulated.

3. Insertion of Recombinant DNA into the Host Cell/Organism

• Transformation: Introducing the recombinant DNA into competent host cells.
• Methods:
  • Heat shock method.
  • Electroporation.
  • Microinjection (directly injecting DNA into cells).

4. Obtaining the Foreign Gene Product

• Expression Systems: Using host cells (bacteria, yeast, plants, or animal cells) to produce the desired protein from the inserted gene.
• Monitoring Expression: Techniques such as Western blotting or ELISA to confirm the production of the protein.

5. Downstream Processing

• Purification: Isolating the desired product from the host cells and other cellular components.
• Characterization: Analyzing the product for its structure and function.
• Formulation: Preparing the product for storage and use.