Principles of Biotechnology
- Biotechnology: Deals with techniques of using live organisms or enzymes to produce products and processes useful to humans.
- European Federation of Biotechnology (EFB) Definition: The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services.
- Core Techniques:
- Genetic Engineering: Techniques to alter the chemistry of genetic material (DNA/RNA), introduce these into host organisms, and change the host's phenotype 1996.
- Bioprocess Engineering: Maintenance of sterile ambience to enable growth of only the desired microbe/eukaryotic cell in large quantities for the manufacture of biotechnological products.
The Conceptual Development of the Principles of Genetic Engineering
- Genetic engineering aims to isolate and introduce only desirable genes into a target organism, overcoming the limitation of traditional hybridisation which includes undesirable genes 2003.
- Origin of Replication (ori): A specific DNA sequence in a chromosome responsible for initiating replication. An alien DNA must be linked to this sequence to multiply in the host NEET 2020.
- Cloning: Making multiple identical copies of any template DNA.
- First Recombinant DNA: Constructed by Stanley Cohen and Herbert Boyer (1972). They isolated an antibiotic resistance gene and linked it to the native plasmid of Salmonella typhimurium.
- Core Mechanism:
- Restriction Enzymes: Act as 'molecular scissors' to cut DNA at specific locations 1998 NEET 2015.
- DNA Ligase: Acts on cut DNA molecules and joins their ends to create recombinant DNA 1998.
- Vector: The plasmid acts as a vector to deliver the alien DNA into the host (e.g., E. coli).
Tools of Recombinant DNA Technology
Enzymes
- Lysing Enzymes: Used to break open cells to release DNA.
- Bacteria: Lysozyme.
- Plant cells: Cellulase/Pectinase.
- Fungal cells: Chitinase 2013 2022 Re.
- Cleaving Enzymes (Nucleases):
- Exonucleases: Remove nucleotides from the ends of the DNA.
- Endonucleases: Make cuts at specific positions within the DNA 2001 NEET 2020.
- Restriction Endonucleases (RE):
- Obtained from bacteria, where they fragment viral DNA as a defense mechanism against bacteriophages 2004.
- First discovered RE: Hind II (recognizes a specific sequence of 6 base pairs) 2020 Covid 2024.
- Palindromic Nucleotide Sequence: REs inspect the length of a DNA sequence and bind to a specific recognition site, cutting the sugar-phosphate backbone on both strands NEET 2019 2020. A palindrome reads the same on both strands in the 5'→3' direction (e.g., 5'-GAATTC-3' and 3'-CTTAAG-5' for EcoRI) 2011 2012 NEET 2020 2024.
- Mode of Cutting:
- Sticky ends: RE cuts a little away from the centre of the palindrome, leaving overhanging single-stranded stretches NEET 2022 Re 2024. Facilitates the action of DNA ligase.
- Blunt ends: Cuts exactly in the centre. (e.g., EcoRV) NEET-II 2016.
- Crucial Rule: To form a recombinant plasmid, both the foreign DNA and the vector MUST be cut by the same restriction endonuclease NEET-II 2016 2024 Re.
Separation and Isolation of DNA Fragments (Gel Electrophoresis)
- Gel Electrophoresis: Technique to separate DNA fragments based on size 2008 2022 Re.
- Matrix: Agarose (a natural polymer extracted from seaweeds) 2011 2022 Re.
- Mechanism: DNA fragments are negatively charged and move towards the anode (+ve electrode) under an electric field NEET 2017 Odisha 2019 2022 Re.
- Size Criterion: The smaller the fragment size, the farther it moves through the sieving agarose gel NEET 2017 Odisha 2019 2024 Re.
- Visualisation: Separated fragments must be stained with ethidium bromide and exposed to UV radiation, appearing as bright orange bands NEET 2017 2020 2021 2023 2024 Re. Exception/Detail: Pure DNA cannot be visualised directly under UV or visible light without staining Odisha 2019 2022.
- Elution: The step where separated bands of DNA are cut out from the agarose gel and extracted 2022.
Cloning Vectors
Plasmids (extra-chromosomal circular DNA) and bacteriophages act as vehicles to deliver alien DNA into host organisms 2000 2001.
Required Features of a Vector
- Origin of Replication (ori): Initiates replication and controls the copy number of the linked DNA NEET 2020 2024.
- Selectable Marker: Helps identify and eliminate non-transformants and selectively permits the growth of transformants. (e.g., antibiotic resistance genes for ampicillin, tetracycline) Odisha 2019.
- Cloning Sites (Restriction sites): Vector needs a recognition site for the RE to link alien DNA. Crucial detail: Ligation at a restriction site within an antibiotic resistance gene (e.g., tetR) results in insertional inactivation, leading to loss of resistance (e.g., loss of tetracycline resistance if ligated at BamHI or SalI sites in pBR322) 2023 Manipur 2024 Re.
- Insertional Inactivation (Alternative Method): Insertion of recombinant DNA within the coding sequence of an enzyme (e.g., β-galactosidase). Recombinant colonies do not produce color and appear white, while non-recombinants produce blue colonies in the presence of a chromogenic substrate NEET 2013.
Vectors for Cloning Genes in Plants and Animals
- Agrobacterium tumefaciens: A natural pathogen of dicots. Delivers T-DNA to transform normal plant cells into tumors (crown gall) 2003 2004 2006 2015.
- Ti Plasmid: The tumor-inducing plasmid of Agrobacterium has been modified into a cloning vector (disarmed) to deliver desirable genes into plants 2004 2024.
- Retroviruses: Disarmed retroviruses are used to deliver desirable genes into animal cells 2010.
Competent Host (Transformation Methods)
- Chemical Treatment: Treating bacteria with divalent cations (e.g., calcium) followed by a heat shock (42°C) enables them to take up recombinant DNA.
- Micro-injection: Recombinant DNA is directly injected into the nucleus of an animal cell.
- Biolistics / Gene Gun: Plant cells are bombarded with high-velocity micro-particles of gold or tungsten coated with DNA 2012 Mains 2012 2023.
Processes of Recombinant DNA Technology
Isolation of DNA
- Cells are treated with lysing enzymes. RNA is removed by ribonuclease; proteins by protease.
- Purified DNA precipitates out after the addition of chilled ethanol Karnataka NEET 2013 NEET 2019 2023.
- Collected as fine threads via spooling 2020 Covid.
Amplification of Gene of Interest using PCR
- PCR (Polymerase Chain Reaction): Used for DNA amplification (~1 billion times).
- Steps in Order NEET 2018 2024 Re:
- Denaturation: Heating to ~94°C to separate double-stranded DNA into single strands Mains 2012 2021.
- Annealing: Two sets of primers attach to the 3' ends of the single-stranded DNA Mains 2012.
- Extension: DNA polymerase synthesizes the DNA strand.
- Enzyme: Taq polymerase, isolated from the thermophilic bacterium Thermus aquaticus. Crucial detail: It is thermostable and remains active during the high-temperature denaturation step 2012 NEET-I 2016 2023 Manipur.
Obtaining the Foreign Gene Product and Downstream Processing
- Bioreactors: Large vessels (100-1000 litres) used to process large volumes of culture to produce recombinant proteins on an industrial scale 2010 NEET 2019 2020 Covid.
- Provide optimal growth conditions (temp, pH, substrate, oxygen).
- Stirred-tank Bioreactors: Facilitate mixing and ensure availability of oxygen throughout the process 2010 NEET-II 2016. They possess an agitator system, oxygen delivery system, and foam control system 2024.
- Downstream Processing: The process of separation and purification of the expressed protein before marketing as a finished product NEET 2017.
- Crucial detail: Gene expression is an upstream process and is NOT a component of downstream processing NEET-II 2016 2020 Covid.