Immunoblotting Techniques | Elisa | Microbial Genetics | Mutation | Unit 4 Biotechnology 6th Sem

The lecture begins by clarifying the syllabus structure of Unit 4, noting that immunoblotting techniques, ELISA, Western Blotting, and Southern Blotting are the main topics. The instructor clarifies that immunoblotting is another name for Western Blotting, and these are all types of blotting techniques used to identify DNA, RNA, or proteins — Southern Blotting for DNA, Northern Blotting for RNA, and Western Blotting for proteins.

ELISA (Enzyme-Linked Immunosorbent Assay) is covered in great detail. The instructor explains it as a highly specific, sensitive, and rapid technique for detecting antigens or antibodies using enzyme-linked antibodies. Four types are covered: (1) Direct ELISA detects antigens by coating them on a microtitre plate and binding with enzyme-linked primary antibody; (2) Indirect ELISA detects antibodies using a known antigen and a secondary enzyme-linked antibody; (3) Sandwich ELISA uses capture and detection antibodies flanking the antigen for higher sensitivity; (4) Competitive ELISA uses labeled antigen competing with sample antigen, where less color development indicates more sample antigen.

Blotting techniques are then explained. Southern Blotting involves DNA isolation, restriction endonuclease digestion, gel electrophoresis for size separation, denaturation to single-stranded DNA, transfer to nitrocellulose membrane, and hybridization with a labeled DNA probe. Western Blotting involves SDS-PAGE separation of proteins, electrical transfer to membrane, blocking, incubation with primary antibody, secondary enzyme-linked antibody, and substrate addition for color detection.

The genetic organization of prokaryotes versus eukaryotes is compared. Prokaryotes have a single circular chromosome, no true nucleus (nucleoid region), generally lack histones and introns, are haploid, and contain plasmids. Eukaryotes have multiple linear chromosomes, a true nucleus with membrane, histone proteins, both exons and introns, and are diploid.

Microbial genetics covers three DNA transfer mechanisms: Transformation (uptake of naked DNA from surroundings, demonstrated by Griffith's experiment with S and R strains of Streptococcus pneumoniae), Transduction (bacteriophage-mediated gene transfer — generalized via lytic cycle and specialized via lysogenic cycle), and Conjugation (direct cell-to-cell contact via sex pilus, involving F+/F- bacteria, Hfr conjugation, and F' conjugation).

Plasmids are reviewed as extrachromosomal circular DNA in bacteria, including fertility, resistance, Col, degradative, and virulence plasmids. Transposons ('jumping genes') are mobile genetic elements that can move within a genome, discovered by Barbara McClintock. They include insertion sequences, composite transposons, and non-composite transposons, and move via cut-and-paste or copy-and-paste mechanisms.

Microbial biotransformation covers enzymatic conversion of compounds by microorganisms, including oxidation, reduction, hydrolysis, hydroxylation, dehydrogenation, methylation, demethylation, isomerization, acetylation, and esterification reactions, with pharmaceutical, environmental, food, and agricultural applications.

Mutation is defined as a sudden, permanent, heritable change in genetic material. Gene mutations include point mutations (transitions and transversions; silent, missense, and nonsense) and frameshift mutations (insertions and deletions). Chromosomal mutations include deletion, duplication, inversion, and translocation. Mutants are classified as morphological, lethal, conditional lethal, nutritional, and biochemical. Mutagens are physical (UV, X-rays), chemical (nitrous acid, mustard gas), or biological (viruses, transposons).