Summary

Principles of Biotechnology: The two core techniques are genetic engineering (altering DNA) and bioprocess technology (growing modified organisms). Restriction enzymes cut DNA at specific palindromic sequences; e.g., EcoRI cuts between G and A in the sequence 5-prime-GAATTC-3-prime, leaving sticky ends. DNA ligase seals these fragments.

Tools of Recombinant DNA Technology: Three essential tools:

• (i) Restriction enzymes (molecular scissors) – produce sticky or blunt ends
• (ii) Cloning vectors (e.g., plasmids, bacteriophages) – must have an origin of replication (ori), selectable marker (e.g., antibiotic resistance gene), and multiple cloning sites (MCS)
• (iii) Host organisms (e.g., E. coli) – competent to take up recombinant DNA

Processes of Recombinant DNA Technology: Steps in order: (1) Isolation of genetic material (DNA) from donor cells; (2) Cutting DNA with restriction enzymes to generate fragments; (3) Amplifying the gene of interest using PCR (polymerase chain reaction) – repeated cycles of denaturation (separating the DNA strands by heat), primer annealing, and extension (a thermostable Taq polymerase copies each strand), producing about a billion copies of the target sequence; (4) Ligation of the gene into a vector using DNA ligase; (5) Insertion of recombinant vector into host cell (transformation); (6) Selection of transformed cells using a marker (e.g., blue-white screening or antibiotic resistance); (7) Obtaining the foreign gene product (expression) and its purification.

Vectors and Their Features: Plasmids are extrachromosomal, self-replicating circular DNA. Key features: ori – controls copy number; selectable marker – helps identify transformants (e.g., ampR, tetR); cloning site – where foreign DNA is inserted. Example: pBR322 – a widely used E. coli plasmid with known restriction sites.

Competent Host and Methods of Gene Transfer: Host cells are made competent (able to take up DNA) by:

• (i) Chemical method – treating with calcium chloride (CaCl2) and heat shock (42°C)
• (ii) Biolistics (gene gun) – DNA-coated gold/tungsten particles shot into cells
• (iii) Microinjection – direct injection into nucleus …