Learning Outcomes:
- Understand the basic principles and processes of biotechnology.
- Identify the tools used in recombinant DNA technology.
- Grasp the methods involved in creating genetically modified organisms.
- Learn about DNA isolation, gene cloning, and recombinant DNA construction.
Biotechnology, in its modern form, involves using living organisms or their enzymes to produce useful products. It encompasses processes like making curd, bread, or wine, which rely on microbes. However, biotechnology now mainly refers to methods involving genetically modified organisms. Techniques like in vitro fertilization, DNA vaccine development, or gene therapy are prominent examples.
Important Concept:
Modern biotechnology is grounded in two key techniques: genetic engineering and bioprocess engineering.
Several tools are essential for recombinant DNA technology. They enable the creation of genetically modified organisms. These tools include restriction enzymes, ligases, vectors, and host organisms.
Restriction enzymes are crucial for cutting DNA at specific sequences. They were first discovered in 1963. Hind II was the first to be characterized for its ability to cut at a specific sequence of six base pairs. Today, over 900 restriction enzymes are known, each cutting at specific sites. The naming convention involves the genus, species, and strain of the bacteria they are derived from.
Important Concept:
Palindromic sequences in DNA read the same in opposite directions, such as 5′ – GAATTC – 3′ and 3′ – CTTAAG – 5′.
Vectors are carriers for foreign DNA fragments. They replicate independently in bacterial cells, and commonly used vectors include plasmids and bacteriophages.
Note:
The insertion of foreign DNA into a cloning vector at a site like BamH I inactivates one antibiotic resistance gene while retaining another, enabling the selection of recombinants.
DNA is hydrophilic, so it cannot pass through cell membranes without assistance. Bacterial cells are made competent to take up DNA by treatment with calcium ions. The process involves heat shock to enhance DNA uptake.
Recombinant DNA technology follows a series of well-defined steps, from DNA isolation to gene amplification and insertion into host organisms. Each step is essential for creating and multiplying recombinant DNA.
DNA, the genetic material in all organisms, must be extracted in its pure form. Cells are broken open using enzymes like lysozyme (for bacteria), cellulase (for plants), or chitinase (for fungi). Once isolated, proteins are removed using proteases, and RNA is degraded with ribonucleases. Chilled ethanol helps precipitate DNA, which is collected as fine threads.
Important Concept:
Pure DNA is essential for subsequent cutting and manipulation steps in recombinant DNA technology.
DNA is cut using restriction enzymes under optimal conditions. This creates fragments, which are separated using gel electrophoresis. DNA fragments, which are negatively charged, move towards the anode in an electric field. The smaller fragments move faster through the agarose gel.
Polymerase Chain Reaction (PCR) amplifies the gene of interest by synthesizing multiple copies of DNA. PCR uses two primers and the enzyme DNA polymerase to replicate DNA. The process involves three main steps: denaturation, primer annealing, and extension.
Table: Comparison of Gene Amplification Techniques
Method Purpose Enzyme Used Amplification Result PCR Amplify specific DNA Thermostable DNA polymerase Billions of DNA copies Cloning Insert gene into vector DNA Ligase Multiple clones in host
After amplification, recombinant DNA is introduced into host cells, which are made competent. For example, if the recombinant DNA confers ampicillin resistance, the transformed cells will survive on ampicillin-containing plates, allowing selection of transformed cells.
Once inside a host, the foreign DNA gets expressed to produce a desirable protein. The cells containing the recombinant DNA are cultured on a large scale. This is achieved through bioreactors, which provide optimal growth conditions.
Table: Bioreactor Types and Features
Bioreactor Type Description Features Stirred-Tank Bioreactor Cylindrical with curved base Stirring system for even mixing Sparged Bioreactor Air bubbled through the culture Better oxygen availability
After biosynthesis, the product must be purified and processed. This includes separation and purification steps, collectively known as downstream processing. The purified product is then formulated with preservatives and subjected to quality control.
Note:
Understanding the intricacies of DNA manipulation has led to significant advancements in biotechnology. The creation of recombinant DNA, gene cloning, and the ability to amplify and express foreign genes have revolutionized modern science and medicine.
What is the purpose of the origin of replication (ori) in a cloning vector?
A) To allow the foreign DNA to replicate in the host cells
B) To introduce antibiotic resistance
C) To insert foreign DNA into host cells
Answer: A