Biotechnology : Principles and Processes

The One Thing

Biotechnology uses living organisms, cells, enzymes, and biological processes to produce useful products and services. Recombinant DNA technology achieves this by isolating, cutting, joining, amplifying, transferring, selecting, and expressing DNA in suitable host cells.

Reactions, Processes and Experiments

Key Terms

• Biotechnology: The use of organisms, cells, enzymes, or biological processes to make useful products and services.
• Genetic engineering: The deliberate modification of an organism's DNA using laboratory techniques.
• Recombinant DNA (rDNA): A DNA molecule formed by joining genetic material from two or more different sources.
• Restriction endonuclease: An enzyme that cuts DNA at specific recognition sequences, often producing sticky ends.
• Recognition sequence: A specific nucleotide sequence identified and cut by a restriction enzyme; many are palindromic sequences.
• Sticky ends: Short, single-stranded DNA overhangs formed by staggered cutting, allowing complementary DNA fragments to pair.
• DNA ligase: An enzyme that joins DNA fragments by forming phosphodiester bonds between adjacent nucleotides.
• DNA polymerase: An enzyme that synthesises a new DNA strand using a template strand.
• Vector: A DNA molecule, such as a plasmid or bacteriophage, used to carry foreign DNA into a host cell.
• Plasmid: A small, circular, self-replicating DNA molecule commonly found in bacteria and used as a cloning vector.
• Origin of replication: A DNA sequence in a vector from which replication begins and that helps determine the copy number of the inserted DNA.
• Selectable marker: A gene that helps identify host cells containing the desired recombinant DNA, often by providing resistance to an antibiotic.
• Cloning site: A specific location in a vector where foreign DNA is inserted using a restriction enzyme.
• Insertional inactivation: A method of identifying recombinants in which insertion of foreign DNA disrupts a marker gene and changes an observable property.
• Competent cells: Host cells treated so that they can take up foreign DNA from their surroundings.
• Transformation: The introduction and uptake of recombinant DNA by a host cell.
• PCR: Polymerase Chain Reaction, a technique used to amplify a selected DNA sequence through repeated cycles of denaturation, primer annealing, and extension.
• Primer: A short single-stranded DNA segment that provides a starting point for DNA synthesis during PCR.
• Taq polymerase: A heat-stable DNA polymerase used in PCR, originally obtained from the bacterium Thermus aquaticus.
• Gel electrophoresis: A technique that separates DNA fragments according to size as they move through an agarose gel in an electric field.
• Elution: The process of removing a desired DNA fragment from an agarose gel after separation.
• Bioreactor: A vessel that provides controlled conditions for large-scale growth of cells or microorganisms and production of a biological product.
• Downstream processing: The purification, formulation, quality testing, and packaging steps carried out after production in a bioreactor.

Easily Confused

• Restriction endonuclease and DNA ligase: Restriction endonuclease cuts DNA at specific recognition sequences, whereas DNA ligase joins DNA fragments by forming phosphodiester bonds.
• Sticky ends and hydrogen bonding: Sticky ends are single-stranded DNA overhangs formed by staggered cutting; hydrogen bonding temporarily pairs complementary sticky ends.
• Vector and plasmid: A vector is any DNA molecule used to carry foreign DNA into a host cell; a plasmid is one common type of vector.
• Selectable marker and cloning site: A selectable marker identifies cells containing particular DNA, whereas a cloning site is the location where foreign DNA is inserted.
• Selection and screening: Selection identifies cells that meet a marker-based condition, whereas screening identifies whether the desired insert is present, including through insertional inactivation.
• Competent cells and transformation: Competent cells are prepared to take up foreign DNA; transformation is the actual introduction and uptake of recombinant DNA.
• DNA polymerase and Taq polymerase: DNA polymerase is the general enzyme that synthesises DNA; Taq polymerase is the heat-stable DNA polymerase used in PCR.
• PCR and gel electrophoresis: PCR amplifies a selected DNA sequence, whereas gel electrophoresis separates DNA fragments according mainly to size.
• Elution and gel electrophoresis: Gel electrophoresis separates DNA fragments; elution removes a desired fragment from the agarose gel.
• Bioreactor and downstream processing: A bioreactor supports controlled biological production; downstream processing purifies, formulates, tests, and packages the product afterward.

What Gets Asked

• Reconstructing the stages of recombinant DNA technology: Questions may require the sequence from isolation of genetic material, cutting, amplification, vector insertion, host introduction, selection, expression, and product recovery. Omitting selection or expression loses marks because DNA uptake alone does not establish successful recombinant production.
• Explaining restriction digestion and ligation: Students may need to use the specific EcoRI example, including 5'-GAATTC-3', cutting between G and A on both strands, sticky ends, complementary hydrogen bonding, and DNA ligase formation of phosphodiester bonds.
• Describing a cloning vector: Answers should identify the origin of replication, selectable markers, and suitable restriction sites. A plasmid is useful because it can replicate independently, carry selectable markers, and accept inserted DNA.
• Describing PCR: Questions may test the required components and the three stages: denaturation at about 94–95 degrees Celsius, primer annealing around 50–65 degrees Celsius, and extension at about 72 degrees Celsius. The amplification relationship after n cycles is approximately 2^n times the starting amount.
• Interpreting gel electrophoresis: Students may be asked to explain why DNA moves towards the positive electrode and why smaller fragments move faster and farther. The negative charge arises from DNA phosphate groups.
• Explaining industrial production: Questions may distinguish bioreactor conditions from downstream processing. Bioreactors control temperature, pH, oxygen supply, agitation, nutrient availability, and foam formation, whereas downstream processing purifies, formulates, quality-tests, and packages the product.