Advances in biotechnology have transformed diagnostics, genetics, and industrial microbiology. From highly sensitive immunoassays to microbial gene transfer and biotransformation, modern techniques allow scientists to analyze, modify, and utilize biological systems with remarkable precision. Unit 4 focuses on essential laboratory techniques, genetic organization, microbial genetics, and mutation—core concepts that underpin pharmaceutical research and biotechnological applications.
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Immunoblotting Techniques: Powerful Tools for Biomolecular Detection
Enzyme-Linked Immunosorbent Assay (ELISA)
ELISA is a widely used immunological technique for detecting and quantifying antigens or antibodies using enzyme-linked reactions. The method relies on antigen–antibody specificity and color development to indicate the presence of a target molecule.
ELISA is extensively applied in disease diagnosis, vaccine development, hormone estimation, and quality control of biological products due to its sensitivity, simplicity, and scalability.
Western Blotting
Western blotting is used to detect specific proteins in a complex biological sample. Proteins are separated by gel electrophoresis, transferred onto a membrane, and probed with specific antibodies.
This technique is invaluable in confirming protein expression, diagnosing viral infections such as HIV, and validating results obtained from ELISA.
Southern Blotting
Southern blotting focuses on DNA detection. DNA fragments are separated, transferred to a membrane, and hybridized with labeled probes.
It is primarily used for gene identification, genetic fingerprinting, and detection of gene rearrangements. Together, blotting techniques form the backbone of molecular diagnostics.
Genetic Organization of Prokaryotes and Eukaryotes
Prokaryotic Genetic Organization
Prokaryotic organisms such as bacteria possess a simple genetic structure. Their DNA is circular, double-stranded, and located in the nucleoid region without a nuclear membrane. Genes are often arranged in operons, allowing coordinated expression of functionally related genes.
This simplicity enables rapid replication and adaptability, making bacteria ideal models for genetic manipulation.
Eukaryotic Genetic Organization
Eukaryotic cells contain linear chromosomes housed within a nucleus. DNA is tightly packed around histone proteins forming chromatin. Unlike prokaryotes, eukaryotic genes contain introns and exons, requiring post-transcriptional processing.
This complex organization allows greater regulation of gene expression but also increases susceptibility to genetic disorders.
Microbial Genetics: Mechanisms of Genetic Exchange
Transformation
Transformation involves the uptake of free DNA fragments from the environment by competent bacterial cells. This process allows bacteria to acquire new genetic traits such as antibiotic resistance and metabolic capabilities.
Transduction
Transduction is mediated by bacteriophages that transfer genetic material from one bacterium to another. It plays a major role in horizontal gene transfer and bacterial evolution.
Conjugation
Conjugation requires direct cell-to-cell contact and the presence of a fertility (F) plasmid. Genetic material is transferred through a conjugation bridge, making it one of the most efficient gene transfer mechanisms.
Plasmids and Transposons
Plasmids are extrachromosomal DNA molecules that carry genes for antibiotic resistance or metabolic functions. Transposons, or “jumping genes,” move within and between genomes, contributing to genetic diversity and resistance development.
These elements are crucial tools in recombinant DNA technology.
Microbial Biotransformation: Harnessing Microbes for Chemical Conversion
Concept of Microbial Biotransformation
Microbial biotransformation refers to the chemical modification of compounds using microorganisms or their enzymes. Unlike chemical synthesis, it occurs under mild conditions and offers high specificity.
Applications in Pharmaceuticals
Microbial biotransformation is widely used to produce steroid drugs, antibiotics, vitamins, and chiral intermediates. It enables cost-effective and eco-friendly production of complex molecules that are difficult to synthesize chemically.
Mutation: Driving Genetic Variation and Evolution
Definition and Importance
Mutation is a permanent, heritable change in genetic material. It is a key source of genetic diversity and plays a crucial role in evolution, microbial adaptation, and drug resistance.
Types of Mutations
Point mutations, involving single base changes
Frameshift mutations, caused by insertion or deletion of nucleotides
Missense and nonsense mutations, altering protein structure or terminating translation
Types of Mutants
Mutants may be classified as morphological, biochemical, or resistant mutants. In biotechnology, mutants are deliberately induced to enhance enzyme production, improve fermentation yields, or develop industrial strains with superior traits.