09Class 12 Biology
Biotechnology: Principles and Processes short notes
Short notes on the tools and steps of recombinant DNA technology: restriction enzymes, vectors, PCR and bioreactors.
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Tools
- Restriction enzymes cut DNA at palindromic sequences. EcoRI was among the first, and it leaves sticky ends.
- DNA ligase joins pieces, and is often called molecular glue.
- Vectors carry DNA, including plasmids and bacteriophages.
- A vector needs an origin of replication (ori), selectable markers and cloning sites.
- pBR322 is a common plasmid vector, with ampicillin and tetracycline resistance genes.
Selecting recombinants
- Insertional inactivation: a gene inserted into a resistance gene stops that resistance.
- With the lacZ system, non-recombinants give blue colonies, and recombinants give white ones.
- Agrobacterium tumefaciens uses its Ti plasmid to insert DNA in plants.
Process
- Isolate DNA by breaking cells, using lysozyme for bacteria, cellulase for plants and chitinase for fungi.
- DNA is precipitated with chilled ethanol.
- Gel electrophoresis separates fragments by size, and smaller ones move farther. Ethidium bromide makes bands visible under UV.
- PCR steps: denaturation, annealing and extension, using heat-stable Taq polymerase.
- Host cells take up DNA by heat shock with calcium ions, microinjection, a gene gun or a disarmed virus.
- Bioreactors grow products at scale, and downstream processing purifies them.
Where students slip up
- Saying larger fragments move farther. Smaller ones do.
- Reversing the colony colours. Recombinants are white, because lacZ is disrupted.
- Forgetting Taq polymerase was chosen since it survives high temperature.
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