Biotechnology - Principles and processes

The One Thing

Biotechnology applies living organisms, cells, enzymes, and genetic material to produce useful products and processes. Genetic engineering achieves this by precisely isolating, modifying, joining, transferring, replicating, selecting, and expressing DNA sequences.

Reactions, Processes and Experiments

Key Terms

• Biotechnology: The application of biological organisms, cells, enzymes, or genetic material to develop useful products and processes.
• Genetic engineering: The deliberate modification of an organism's DNA by adding, removing, or altering specific genes.
• Recombinant DNA: A DNA molecule formed by joining genetic material from two 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 particular 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 them.
• DNA polymerase: An enzyme that synthesises a new DNA strand using a template strand.
• Vector: A DNA molecule used to carry a foreign gene into a host cell, such as a plasmid or bacteriophage.
• Plasmid: A small, circular, independently replicating DNA molecule commonly found in bacteria and used as a cloning vector.
• Origin of replication: The DNA sequence in a vector from which replication begins; it helps determine the copy number of the inserted DNA.
• Selectable marker: A gene that helps identify host cells containing the recombinant vector, often by providing resistance to a particular antibiotic.
• Cloning site: A specific location in a vector where foreign DNA can be inserted using a restriction enzyme.
• Insertional inactivation: A method of identifying recombinant cells because insertion of foreign DNA disrupts the function of a marker gene.
• 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.
• Polymerase chain reaction: A technique used to amplify a selected DNA segment through repeated cycles of denaturation, primer binding, and extension.
• Denaturation: Separation of the two DNA strands by heating during PCR.
• Annealing: Binding of short DNA primers to complementary sequences on the separated template strands.
• Extension: Synthesis of new DNA strands by a thermostable DNA polymerase from the primers.
• Gel electrophoresis: A technique that separates DNA fragments according to size as they move through an agarose gel in an electric field.
• Gene cloning: Production of many identical copies of a selected gene or DNA fragment.
• Expression vector: A vector containing regulatory sequences that enable the inserted gene to be transcribed and translated in a host cell.
• Bioreactor: A vessel designed to provide controlled conditions for large-scale growth of cells or microorganisms and production of biological products.
• Downstream processing: The recovery, purification, formulation, and quality testing of a product after it has been produced in a bioreactor.
• Promoter: A regulatory DNA sequence where RNA polymerase begins transcription; a strong, suitable promoter can increase gene expression.
• Palindromic sequence: A DNA sequence that reads the same in the 5' to 3' direction on both complementary strands.

Easily Confused

• PCR and recombinant DNA technology: PCR rapidly amplifies a selected DNA region, whereas recombinant DNA technology inserts and maintains that region in a vector or host.
• Gene cloning and reproductive cloning: Gene cloning produces many identical copies of a DNA fragment, whereas reproductive cloning produces an entire organism.
• Molecular, cellular, and reproductive cloning: Molecular cloning copies DNA, cellular cloning copies cells, and reproductive cloning produces an entire organism.
• Restriction enzymes and DNA ligase: Restriction enzymes cut DNA at specific sites and may create compatible ends; DNA ligase joins DNA fragments permanently.
• Recognition sequences and sticky ends: A recognition sequence is the DNA site identified by a restriction enzyme; sticky ends are the single-stranded overhangs produced by staggered cutting.
• Competent cells and transformation: Competent cells are prepared to take up DNA; transformation is the actual introduction and uptake of recombinant DNA.
• Origin of replication and promoter: The origin of replication controls initiation of vector replication and contributes to copy number, whereas the promoter controls initiation of transcription.
• Selectable marker and insertional inactivation: A selectable marker identifies cells containing a vector, whereas insertional inactivation identifies recombinant cells when inserted DNA disrupts a marker gene.
• Transformation methods: Calcium ion treatment followed by heat shock and electroporation are methods for making bacterial cells take up DNA; microinjection, chemical methods, and electroporation are methods used for animal cells.
• Expression and downstream processing: Expression produces the biological product in the host cell; downstream processing recovers, purifies, formulates, and tests it.

What Gets Asked

• Describe the stages of recombinant DNA technology. Marks are lost by omitting selection of transformants, gene expression, or downstream processing after DNA insertion.
• Explain how restriction enzymes and DNA ligase work together. The distinction must be maintained: restriction enzymes create compatible DNA ends, while ligase forms phosphodiester bonds to join the fragments.
• Interpret the EcoRI example and DNA palindromes. The sequence 5'-GAATTC-3' and the definition of reading the same in the 5' to 3' direction on both complementary strands must be retained.
• Explain PCR and calculate amplification. The three stages—denaturation, annealing, and extension—must be given in order, and the equation N = N0 x 2^n must not be confused with actual amplification, which is lower in practice.
• Identify the components and requirements of a plasmid vector. Answers should include the origin of replication, selectable marker, cloning site, and, where relevant, a promoter; the inserted gene must also be in the correct orientation and reading frame for efficient expression.
• Explain large-scale biotechnological production. A complete answer must include bioreactor control of temperature, pH, oxygen supply, agitation, nutrient concentration, foam, and contamination, followed by downstream processing and quality testing.