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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.