Chapter at a glance
- Biotechnology deals with techniques of using live organisms or enzymes from organisms to produce products and processes useful to humans; in the restricted sense it refers to processes using genetically modified organisms on a larger scale.
- The two core techniques enabling modern biotechnology are genetic engineering (techniques to alter the chemistry of genetic material, introduce it into host organisms and change the phenotype) and bioprocess engineering (maintenance of sterile ambience in chemical engineering processes to grow only the desired microbe/eukaryotic cell in large quantities).
- Construction of recombinant DNA requires linking an alien piece of DNA with the origin of replication so that the alien DNA can replicate and multiply in the host; this is called cloning.
- Three basic steps in genetically modifying an organism: (i) identification of DNA with desirable genes, (ii) introduction of the identified DNA into the host, (iii) maintenance of introduced DNA in the host and transfer to its progeny.
- Restriction endonucleases act as molecular scissors that cut DNA at specific palindromic recognition sequences, producing sticky ends that facilitate ligation by DNA ligase.
- Cloning vectors (engineered plasmids or bacteriophages) must possess ori (origin of replication), selectable marker(s) and preferably single cloning sites; selectable markers enable differentiation of recombinants from non-recombinants (e.g., antibiotic-resistance genes or insertional inactivation of β-galactosidase).
- Recombinant DNA technology processes include isolation of DNA, fragmentation by restriction endonucleases, isolation of desired fragment, ligation into vector, transfer into host, large-scale culture in bioreactors and downstream processing.
- PCR amplifies the gene of interest in vitro using thermostable DNA polymerase (Taq polymerase from Thermus aquaticus), primers and repeated cycles of denaturation, primer annealing and extension.
Definitions and laws
- Biotechnology (EFB definition): “The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services.”
- Origin of replication (ori): A specific DNA sequence responsible for initiating replication; any alien DNA linked to it can replicate and multiply in the host organism.
- Selectable marker: A gene (usually antibiotic-resistance gene) that helps in identifying and eliminating non-transformants and selectively permitting the growth of transformants.
- Cloning: The ability to multiply copies of any template DNA (alien piece of DNA) in an organism after it becomes part of a chromosome that has an origin of replication.
- Recombinant DNA: A new combination of circular autonomously replicating DNA created in vitro by linking a gene (e.g., antibiotic-resistance gene) with a plasmid vector using DNA ligase.
- Palindromic nucleotide sequence: A sequence of base pairs that reads the same on the two strands when the orientation of reading is kept the same (e.g., 5′-GAATTC-3′ / 3′-CTTAAG-5′).
- Downstream processing: Series of processes (separation, purification, formulation with preservatives, quality control) after the biosynthetic stage to obtain the finished product ready for marketing.
Important diagrams and activities
- Figure 9.1: Steps in formation of recombinant DNA by action of restriction endonuclease EcoRI — demonstrates recognition of palindromic sequence, cutting to produce sticky ends and role of ligase.
- Figure 9.2: Diagrammatic representation of recombinant DNA technology — shows overall process from cutting source and vector DNA to obtaining recombinant molecule.
- Figure 9.3: Agarose gel electrophoresis showing migration of undigested and digested DNA fragments — demonstrates separation of DNA fragments according to size under electric field.
- Figure 9.4: E. coli cloning vector pBR322 showing restriction sites, ori and antibiotic-resistance genes (ampR, tetR) — illustrates features required in a cloning vector and insertional inactivation.
- Figure 9.5: DNA precipitation by chilled ethanol and spooling — shows isolation of pure DNA.
- Figure 9.6: Polymerase chain reaction (PCR) cycle — depicts the three steps (denaturation, primer annealing, extension) repeated to amplify DNA.
- Figure 9.7: Simple stirred-tank bioreactor and sparged stirred-tank bioreactor — illustrates components (agitator, oxygen delivery, temperature/pH control, foam control, sampling ports) for large-scale culture.
Common misconceptions and exam pitfalls
- Confusing traditional biotechnology (curd, bread, wine) with modern biotechnology that specifically uses genetically modified organisms and recombinant DNA technology.
- Assuming any DNA fragment introduced into a cell will automatically replicate; it must be linked to ori.
- Mixing up exonucleases (remove nucleotides from ends) with endonucleases (cut at specific internal sites).
- Forgetting that restriction enzymes cut a little away from the centre of the palindrome, leaving sticky ends, or that same enzyme must be used on both vector and source DNA.
- Overlooking that selectable markers work by insertional inactivation or by differential growth on antibiotic plates; students often miss the two-plate selection method or β-galactosidase colour selection.
- Thinking PCR uses any DNA polymerase; it requires thermostable Taq polymerase to withstand denaturation temperature.
Formula sheet
No numerical formulas or equations are present in the chapter.