BIOTECHNOLOGY: PRINCIPLES AND PROCESSES

Genetic engineering = creation of recombinant DNA; gene cloning; gene transfer — overcomes limitations of traditional hybridisation.

First recombinant DNA — Stanley Cohen & Herbert Boyer; 1972; antibiotic resistance gene + native plasmid of Salmonella typhimurium.

Cloning = ability to multiply copies of antibiotic resistance gene in E. coli.

Plasmid = autonomously replicating circular extra-chromosomal DNA.

Plasmids are autonomously replicating DNA and are extrachromosomal DNA — both statements are correct.

Three basic steps — identification → introduction → maintenance & transfer to progeny.

The enzymes essential for gene cloning are restriction enzymes, DNA ligase and DNA polymerase; DNA mutase and DNA recombinase are NOT essential for gene cloning.

First restriction endonuclease = Hind II; recognises specific sequence of 6 base pairs.

Exonucleases = remove nucleotides from ends; Endonucleases = cut at specific positions within DNA.

Restriction endonucleases are called molecular scissors, are the enzymes responsible for restricting the growth of bacteriophages in E. coli, and recognise specific palindromic base-pair sequences.

Restriction endonucleases do NOT cut DNA only at the centre of palindromic sites — they cut at specific positions within or near the palindrome, producing either sticky or blunt ends.

Removing nucleotides only from the ends of DNA fragments is the action of EXONUCLEASES, not of restriction endonucleases.

Palindromic sequence — 5′-GAATTC-3′ / 3′-CTTAAG-5′; reads same on both strands in same orientation.

Sticky ends = cut away from centre of palindrome; Blunt ends = cut at centre.

Stickiness of ends facilitates action of DNA ligase.

DNA = negatively charged → moves toward anode; Agarose = natural polymer from sea weeds.

Ethidium bromide staining → UV radiation → bright orange bands.

Elution = cutting out & extracting separated DNA bands from agarose gel.

DNA is cut by molecular scissors and the resulting fragments separate according to their size in an agarose gel upon electrophoresis.

Separated DNA fragments CANNOT be seen without staining, and ethidium bromide-stained DNA is seen under UV light — NOT in visible light.

Smaller DNA fragments are observed near the anode while larger fragments remain near the wells in an agarose gel.

DNA fragments extracted from gel electrophoresis (elution) can be used in the construction of recombinant DNA.

ori = controls copy number; Selectable marker = identifies transformants; Cloning sites = preferably single restriction site.

Match-the-list: Transformation ↔ transfer of DNA to host bacteria; Cloning site ↔ restriction enzyme recognition sequence; Selection ↔ antibiotic; Ori ↔ replication.

Selectable markers = antibiotic resistance genes (Ampicillin, Chloramphenicol, Tetracycline, Kanamycin).

pBR322 — amp^R & tet^R; restriction sites; insertional inactivation of tet^R at BamH I site.

Foreign DNA at BamH I → loses tetracycline resistance.

Insertion of foreign DNA at the BamH I site of pBR322 results in loss of resistance towards TETRACYCLINE only — the amp^R gene remains intact and is disrupted only at the Pst I site.

Blue colonies = non-recombinant (no insert); White = recombinant (insert present; β-galactosidase inactivated).

Insertional inactivation of β-galactosidase → recombinant colonies = no colour.

Colonies WITHOUT blue colour (white colonies) have the DNA insert in the plasmid and are identified as recombinant colonies; blue colonies are non-recombinant.

When an alien DNA is inserted at the EcoR I site within the lacZ gene, white coloured colonies are selected as the recombinants.

Ti plasmid of Agrobacterium tumifaciens → modified into cloning vector for plants.

Retroviruses in animals → ability to transform normal cells into cancerous cells.

Match-the-list: Genetically modified organism ↔ Bt cotton; Thermostable DNA polymerase ↔ Thermus aquaticus; Ti plasmid ↔ Agrobacterium tumefaciens; pBR322 ↔ Escherichia coli.

Biolistics/Gene gun = micro-particles of Gold or Tungsten coated with DNA → bombard plant cells.

Micro-injection = for animals; Gene gun = for plants.

Know all six steps in correct sequence.

Lysozyme = bacteria; Cellulase = plants; Chitinase = fungus.

Lysozyme is used for disrupting bacterial cells while cellulase is used for plant cells, because isolation of genetic material requires disruption of cells to release DNA from within the membranes.

Purified DNA precipitated by chilled ethanol → fine threads = Spooling.

PCR steps = Denaturation → Annealing → Extension (sequence matters!).

Correct sequence of steps in each PCR cycle = Denaturation → Annealing → Extension.

During PCR, primers bind to the DNA strands in the ANNEALING step.

PCR amplifies DNA following the equation 2ⁿ, where n = number of cycles.

Thermus aquaticus — source of thermostable DNA polymerase (Taq polymerase).

If high temperature is not maintained → Denaturation step is affected first.

Most common bioreactor = stirring type; components listed; provides optimal conditions.

Bioreactors are for large scale, NOT small scale cultures.

The foam breaker is the bioreactor component that disrupts and dissipates foam generated during operation, preventing foam overflow. Learn the labelled diagram: agitator/impeller, sparger, foam breaker, cooling jacket, sampling ports.

Downstream processing = Separation + Purification; preservatives; clinical trials; quality control.

'Expression' is part of upstream processing, NOT downstream.