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Biotechnology
Biotechnology
TECHNOLOGY**
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**Biotechnology: Technological Exploitation of Biological Processes**
**Definition:** Biotechnology is the technological exploitation of biological processes for the benefit of
mankind.
- Hybridization Techniques:
- Genetic Engineering:
- Example: Stanley Cohen & Herbert Boyer (1972) constructed the first recombinant DNA.
- Linked a gene of antibiotic resistance from the plasmid of Salmonella typhimurium with a plasmid
of E. coli and transferred it into E. coli.
**Classical Biotechnology:**
Note: Biotechnology has diverse applications and is constantly evolving, leading to significant
advancements in various fields, including medicine, agriculture, and industry.
- Purified source DNA and vector DNA are cut with a specific restriction enzyme.
- Endonucleases: Cut at specific recognition sequences, often palindromic, leaving sticky ends.
- E.g.: EcoRI
**V. Separation of Desired DNA Fragment Using Gel
Electrophoresis** - 1st letter (capital and italics) - Genus of
the prokaryotic cell (E for Escherichia)
- Cleavage by endonuclease produces many different DNA
fragments. - 2nd & 3rd letters - Species (co for coli)
- Gel electrophoresis separates DNA fragments based on size - 4th letter - Strain name of bacteria (R for
using an electric field. RY 13)
- Smaller fragments move farther through the gel. - Following Roman numerals indicate the
order of isolation from the strain (I for
- DNA bands can be visualized by staining with Ethidium first).
Bromide under UV light.
- Requirements: DNA template, free nucleotides, primers (complementary to 3' end of template), and
DNA polymerase (e.g., Taq polymerase).
- Steps:
- Desired DNA fragments are incorporated into the vector, which has been cut with the same
restriction endonuclease as the source DNA.
- Ligase is used to join the DNA fragments and vector, creating recombinant DNA (rDNA). Desired DNA
+ Vector = rDNA
- Sequence where replication starts, allowing DNA linked to ori to replicate within host cells.
- Vectors that replicate to a number equal to the copy number of the vector when linked with DNA.
- Examples: Antibiotic resistance genes (e.g., ampR, tetR), Enzyme-producing genes (e.g., lacZ gene
producing β-galactosidase).
- Should have at least one recognition site for restriction enzymes to link desired DNA.
- Multiple recognition sites may generate several fragments, complicating gene cloning.
**Types of Vectors:**
1. Plasmids:
- pBR322 features:
- 8 restriction enzyme recognition sites.
- Retroviruses have the ability to transform normal cells into cancerous cells.
3. Bacteriophages:
2. Micro-injection: Injecting DNA directly into the nucleus of an animal cell using a micro-needle.
3. Gene gun method: Coating micro-particles of gold or tungsten with DNA and shooting them at
plant cells with high velocity.
**Selection of Transformants:**
Selection of transformed cells (those carrying the recombinant DNA) from non-transformed ones is
achieved using selectable markers and insertional inactivation. There are two types of selection
methods:
- This method involves insertional inactivation of the lac-Z gene and is known as the Blue-White
selection method.
- Normal bacteria (non-transformants) contain the lac-Z gene on their plasmids, which produces the
enzyme β-galactosidase.
- Transformant bacteria with foreign DNA inserted within the lac-Z gene experience inactivation,
leading to no β-galactosidase production and resulting in white colonies. These are identified as
recombinant colonies.
- Extraction and purification of the desired product from the culture medium is known as Downstream
Processing.
- Procedure:
1. Separation and purification of gene products
2. Addition of suitable preservatives.
3. Goes through clinical trials (in the case of drugs).
4. Quality control testing.