BIOTECHNOLOGY

Bio → means life (from Greek “bios”) → refers to living things like plants,
animals, microorganisms, or any biological material.

Technology → means practical application of scientific knowledge (from

Greek “techne” = skill + “logos” = study).

So, Biotechnology literally means:

The technology of life — using living organisms, their parts, or their
processes for practical purposes, especially to create products or solve
problems.

Example:

Using yeast (bio) + fermentation process (technology) → to make bread,

beer, or wine.
Definition-
The application of scientific and engineering principles for processing of
materials by biological agents to provide goods and services for human
welfare.

History-
Term coined by: Karl Ereky (1919).

Organisation for Economic Co-operation and Development-

Definition: Development and utilization of biological forms, products, or
processes for maximum benefit to humans and other forms of life.

Includes principles from microbiology, genetics, biochemistry, chemical

engineering, mathematics, statistics, computers, and industrial processes.
Two phases of Biotechnology:

1. Traditional Biotechnology:
Ancient use of fermentation for bread, alcohol, cheese, etc.
Microbiologists, chemists, and biochemists later discovered fermentation
processes in detail.

2. Modern Biotechnology:
Includes rDNA technology, polymerase chain reaction,cell culture, fusion,
bioprocessing.

1970: rDNA technology developed.

1973: Demonstrated by Stanley Cohen & Herbert Boyer.
Helps in modifying genetic material for production of new products.
Principles & Processes of Biotechnology-

1. Genetic Engineering
Manipulation of genetic material in a desired and predetermined way in vitro.
Alters phenotype by changing genotype.
Process:
Nucleic acid produced outside an organism and incorporated into host using
vectors (virus, plasmid, etc.).
Host expresses the introduced gene to produce new traits.
Applications: Gene combination, new gene introduction, gene removal/
replacement/repair.

2. Chemical Engineering
Maintenance of sterile environment for production of:
Vaccines, Antibodies, Enzymes, Organic acids, Vitamins, Therapeutic products
Tools Used in Gene Cloning / rDNA Technology

1) Instruments / Devices
Living cells make DNA, RNA, proteins, etc., which differ in size, charge, and
properties.
Separation techniques: electrophoresis, gel filtration, ion-exchange
chromatography, spectroscopy, mass spectrometry.

Electrophoresis: separates charged molecules (DNA, RNA, proteins) under an

electric field; DNA fragments move to the anode (small fragments move faster).
Types: PAGE, SDS-PAGE, agarose gel electrophoresis.

PCR (Polymerase Chain Reaction): amplifies DNA sequence.

2) Biological Tools
(a) Enzymes
Restriction endonucleases (biological scissors) cut DNA at specific sites.
Other enzymes: DNA ligase, reverse transcriptase, DNA polymerase, alkaline phosphatase.

(b) Cloning Vectors

Carrier DNA to transfer desired DNA into host.
Features: low molecular weight, self-replicating, ori gene, restriction sites, marker genes.

Examples: Plasmids (pBR322, pUC), cosmids, bacteriophages (M13, λ phage), BAC, YAC, MAC,
transposons, baculoviruses.

(c) Competent Host

Organism for rDNA cloning.
Common: E. coli; others: Bacillus, Haemophilus, Helicobacter.
3) PCR (Gene Amplification)
In vitro technique for billions of DNA copies.

Invented by Kary Mullis (1985).

Requires:
1. Target DNA

2. Forward & reverse primers

3. dNTPs (dATP, dGTP, dTTP, dCTP) & Mg²⁺

4. Thermostable DNA polymerase (Taq polymerase)

Device: Thermal cycler for automated temperature changes.
PCR Techniques:

1. Each cycle of PCR doubles the number of target DNA.

2. One cycle completes in 3-54 minutes.
3. Thus 20-30 cycles produce about 2 billion copies of DNA
4. PCR is performed by an automated machine.
5. Amplified copies of DNA can be purified by gel electrophoresis
6. The steps involved in PCR are as follow.
7. Initially the DNA segment, deoxyribonucleotide triphosphates and DNA polymerase enzyme
are mixed.

1. Denaturation by Heat
The DNA is heated at 90-98°C which breaks hydrogen bonds and makes ssDNA .
DNA with more G-C bonds requires much higher temperature

2. Annealing
Paining of primers to ssDNA (single standard DNA) is called as annealing.
The temperature is controlled at 40- 60°C
3.Polymerization:
At 70-75°C Taq polymerase adds
dNTPs on ssDNA and produces two
copies of the DNA.
It is known as primer extension. It
takes place in 2 minutes.
Restriction Fragments and Restriction Endonuclease:
1. Restriction: Cutting of DNA strands by restriction enzymes.
2. Restriction Fragments: DNA fragments resulting from
restriction enzyme cuts.
3. Discovery: Smith, Nathan, and Arber discovered restriction
enzymes (Nobel Prize in 1978).
Restriction Endonuclease:
1. Function: Cuts DNA at specific sites.
2. Types:
- Type I: Functions as endonuclease and methylase.
- Type II: Recognizes and cuts DNA at specific palindromic
sites (used in rDNA technology).
- Type III: Cuts DNA at specific non-palindromic sites.
Importance:
Restriction endonucleases are essential for gene cloning and
recombinant DNA technology.
Recognition Sequence:
1. Definition: Specific DNA sequence where restriction endonuclease cuts DNA.
2. Characteristics:
- Recognized by specific restriction enzymes.
- Often palindromic (reads same forward and backward).
- Can produce cohesive/sticky ends or blunt ends.

Key Features:
1. Palindromic sequences: DNA sequences that read the same in both directions.
2. Cohesive/sticky ends: Single-stranded projections that facilitate base pairing.

Example:
EcoRI restriction enzyme recognizes and cuts DNA at specific palindromic
sequences, producing cohesive ends.
Plasmids:
1. Definition: Small, circular, double-stranded, extrachromosomal DNA molecules.
2. Key Features:
- Autonomously replicating.
- Commonly used in recombinant DNA (rDNA) technology.
- Often used as vectors for gene cloning.
Importance:
Plasmids play a crucial role in genetic engineering, allowing for the insertion and
expression of foreign genes in host organisms like E. coli.
Agrobacterium tumefaciens:
1. Soil bacterium: Causes crown gall
disease in plants.

2. Ti plasmid: Tumor-inducing
plasmid with T-DNA transposon.

3. Gene transfer: Used to transfer

genes into plant cells, enabling
genetic engineering.

Applications:
1. Crop improvement: Introducing desirable traits like pest resistance (cry gene) or nitrogen
fixation (Nif gene).
2. Plant biotechnology: Agrobacterium-mediated transformation is a key tool in plant genetic
engineering.Its ability to transfer DNA into plant cells makes it a valuable tool in biotechnology.
Recombinant DNA Technology:
rDNA Technology: A technique to transfer genes between organisms using vectors,
enabling production of desired products.

Steps:
1. Isolation of Desired Gene: Isolate gene from donor organism using restriction
endonuclease.
2. Selection of Vector: Choose a suitable vector (plasmid or virus) and cleave it with
same restriction enzyme.
3. Formation of Recombinant DNA: Join vector and foreign DNA using DNA ligase.
4. Introduction of rDNA into Host: Introduce rDNA into host organism (e.g., bacteria).
5. Selection of Transformed Host: Separate transformed hosts from non-transformed
ones.
6. Multiplication of Transformed Host: Clone transformed hosts to produce multiple
copies.
7. Expression of Gene: rDNA expresses and produces desired product.
Applications of Biotechnology:
4. Industrial Biotechnology:
1. Plant Biotechnology: - Enzyme biotechnology
- Plant tissue culture - Protein engineering
- Genetically modified (GM) plants - Metabolic engineering

2. Animal Biotechnology: 5. Environmental Biotechnology:

- Animal tissue culture - Pollution control
- Gene therapy - Biofuels
- Pharmacogenomics - Bioremediation

3. Microbial Biotechnology: 6. Genomics:

- Biofertilizers - Genomic sequencing
- Biopesticides - Gene function analysis
- Integrated Pest Management (IPM) - Bioinformatics

Biotechnology has diverse applications across various fields, improving

agriculture, healthcare, industry, and the environment.
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