Principles Of Biotechnology

Biotechnology involves using living organisms or their products to create useful human products and processes. While traditional applications like making curd, bread, and wine fall under this umbrella, modern biotechnology focuses on manipulating organisms, particularly using genetically modified organisms (GMOs), for large-scale production. It also encompasses techniques like in vitro fertilization, gene synthesis, DNA vaccines, and gene therapy.

The European Federation of Biotechnology (EFB) defines it as the integration of natural sciences and organisms, cells, parts thereof, and molecular analogues for products and services.

The two core principles of modern biotechnology are:

1. Genetic Engineering: Techniques to alter the chemistry of genetic material (DNA/RNA), introduce it into host organisms, and modify the host's phenotype. This allows for the precise insertion of desirable genes without undesirable ones, overcoming limitations of traditional hybridization.
2. Bioprocess Engineering: Maintaining sterile (contamination-free) environments in chemical engineering processes to facilitate the large-scale growth of desired microbes or eukaryotic cells for producing biotechnological products like antibiotics, vaccines, and enzymes.

The ability to multiply an alien DNA piece requires linking it to an 'origin of replication' (ori) sequence, enabling its replication along with the host DNA. This process of creating multiple identical copies is called cloning.

Tools Of Recombinant Dna Technology

Recombinant DNA technology relies on several key tools:

Restriction Enzymes

These enzymes, discovered in 1963, act as 'molecular scissors' to cut DNA at specific recognition sequences. The first identified was Hind II, which recognizes a six-base pair palindromic sequence. Restriction enzymes are classified as exonucleases (remove nucleotides from ends) and endonucleases (cut within the DNA). They cut the DNA strands at specific points within or near the recognition site, often leaving 'sticky ends' – short, single-stranded overhangs that can hydrogen bond with complementary ends. These sticky ends facilitate the joining of DNA fragments using DNA ligase. Examples include EcoRI, which cuts at GAATTC.

DNA fragments are separated based on size using gel electrophoresis. DNA, being negatively charged, moves towards the anode through an agarose gel matrix. Smaller fragments move faster and further. Visualization involves staining with ethidium bromide and exposure to UV light, revealing orange bands.

Cloning Vectors

Cloning vectors are DNA molecules (like plasmids or bacteriophages) that can replicate autonomously within a host organism and carry a foreign DNA segment. Essential features of a cloning vector include:

• Origin of Replication (ori): A sequence that initiates DNA replication and controls the copy number of the linked DNA.
• Selectable Marker: Genes (e.g., antibiotic resistance genes like ampicillin or tetracycline resistance) that help identify and eliminate non-transformed cells, allowing only transformed cells to grow.
• Cloning Sites: Specific recognition sites for restriction enzymes, ideally a single site within a selectable marker gene, to allow insertion of foreign DNA.

Insertional Inactivation: A more efficient method uses selectable markers that produce color. If a foreign DNA is inserted into the coding sequence of an enzyme (like β-galactosidase), it inactivates the enzyme, preventing color formation in the presence of a chromogenic substrate. Colonies without the insert produce blue color, while recombinant colonies remain colorless.

Vectors can also be derived from viruses (retroviruses in animals) and plasmids of Agrobacterium tumefaciens (Ti plasmid) for gene transfer in plants.

Competent Host (For Transformation With Recombinant Dna)

Since DNA is a hydrophilic molecule, it cannot easily pass through cell membranes. Host cells must be made competent to take up foreign DNA. Methods include:

• Calcium Chloride Treatment: Treating bacterial cells with divalent cations like Ca2+ increases membrane permeability. Cells are then incubated with recombinant DNA on ice, followed by brief heating at 42°C (heat shock) and cooling on ice.
• Micro-injection: Recombinant DNA is directly injected into the nucleus of an animal cell.
• Biolistics (Gene Gun): Cells (usually plant cells) are bombarded with high-velocity micro-particles coated with DNA.
• Disarmed Pathogen Vectors: Using modified viruses or bacteria that can deliver recombinant DNA into host cells without causing disease.

Processes Of Recombinant Dna Technology

Recombinant DNA technology involves a series of sequential steps:

Isolation Of The Genetic Material (Dna)

The DNA containing the gene of interest must be isolated in a pure form, free from other macromolecules like RNA, proteins, lipids, and polysaccharides. This involves lysing the cells (using enzymes like lysozyme for bacteria, cellulase for plants, chitinase for fungi) and then removing RNA with ribonuclease (RNase) and proteins with protease. Finally, purified DNA precipitates out upon addition of chilled ethanol.

Cutting Of Dna At Specific Locations

The isolated DNA (both the source DNA containing the gene of interest and the vector DNA) is cut using the same restriction endonuclease. This enzymatic digestion generates DNA fragments with compatible ends (usually sticky ends).

Amplification Of Gene Of Interest Using Pcr

Polymerase Chain Reaction (PCR) is used to amplify multiple copies of the desired gene fragment in vitro. It utilizes two sets of primers (short DNA sequences) and a thermostable DNA polymerase (like Taq polymerase from Thermus aquaticus) that remains active at high temperatures. The process involves cycles of denaturation, primer annealing, and extension, rapidly multiplying the target DNA sequence.

Insertion Of Recombinant Dna Into The Host Cell/Organism

The isolated gene fragment is joined to the cut vector using DNA ligase, forming a recombinant DNA molecule. This recombinant DNA is then introduced into a competent host cell (e.g., bacteria, plant cell, animal cell) using methods like heat shock, micro-injection, biolistics, or disarmed pathogen vectors.

Obtaining The Foreign Gene Product

The host cell containing the recombinant DNA must be cultured under optimal conditions to allow the foreign gene to be expressed, producing the desired protein (recombinant protein). This requires careful control of temperature, pH, nutrients, and oxygen in large-scale culture vessels called bioreactors. The cells are grown in either batch or continuous culture systems to maximize biomass and protein yield.

Downstream Processing

After the biosynthetic stage, the desired product (protein) needs to be separated, purified, and formulated into a finished product. This involves several downstream processing steps, including separation, purification, and quality control testing, tailored to the specific product.

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