MICROFT-C8.pdf

MICROFT C8 Techniques of Recombinant DNA Technology 1. Polymerase Chain Reaction (PCR): The Role of Recombinant DNA Technology in In vitro multiplication o...

MICROFT C8 Techniques of Recombinant DNA Technology 1. Polymerase Chain Reaction (PCR): The Role of Recombinant DNA Technology in In vitro multiplication of specific DNA segments. Biotechnology Steps: Denaturation, priming, extension. Automation: Thermocycler using Taq DNA polymerase. ○ Biotechnology : The use of microorganisms to Variation: Real time PCR for quantifying DNA sequences. make practical products, a practice dating back thousands of years. Applications and Impact ○ Industrial Applications : Acetone, butanol, Industrial and Environmental: Enhanced production antibiotics, paper, textiles, vitamins, environmental processes, bioremediation, resource extraction. Medical: Development of pharmaceuticals, gene therapy, clean up, mining. diagnostics. ○ Modern Advances : Since the 1990s, recombinant Research: Simplified gene isolation, genetic mapping, and DNA technology (genetic engineering) has enabled sequencing. intentional genome modification for practical purposes. Important Concepts ○ Recombinant DNA Technology: A collection of tools Goals of Recombinant DNA Technology and techniques for genome manipulation. 1. Eliminate Undesirable Traits : ○ Genetically Modified Organisms (GMOs): Example: Genes inserted into plants for pest or freeze Organisms with inserted genes from other species. resistance. Example: Gene therapy for severe combined ○ Mutagens and Reverse Transcriptase: Key tools for immunodeficiency (SCID). inducing genetic changes and synthesizing cDNA. 2. Combine Beneficial Traits : ○ Restriction Enzymes and Vectors: Essential for Example: Laboratory animals mimicking human cutting and inserting DNA into host genomes. susceptibility to HIV. 3. Create Organisms for Product Synthesis : ○ PCR: A critical technique for amplifying DNA for Example: Bacteria producing human insulin. various applications. Tools of Recombinant DNA Technology 1. Mutagens : Applications of Recombinant DNA Technology Physical and chemical agents causing mutations. Recombinant DNA technology has a wide range of Example: Mutagens used to develop Penicillium strains applications across various fields, solving problems and producing more penicillin. creating products in research, medicine, and agriculture. 2. Reverse Transcriptase : Converts RNA to complementary DNA (cDNA). Genetic Mapping cDNA lacks noncoding sequences, making it expressible in ○ Genetic Mapping: Locating genes on nucleic acid prokaryotic cells. molecules. Applications: Producing human proteins like growth factor, insulin, blood clotting factors. ○ Utility: Provides insights into an organism’s 3. Synthetic Nucleic Acids : metabolism, growth characteristics, and relatedness In vitro production of DNA/RNA. to other microbes. Uses: Elucidating genetic code, creating specific genes, ○ Example: Discovery of hepatitis G virus, presumed synthesizing probes and antisense molecules, and PCR to cause hepatitis due to its genetic similarity to primers. 4. Restriction Enzymes : known hepatitis viruses. Enzymes that cut DNA at specific sites. Types: Sticky ends (e.g., EcoRI) and blunt ends (e.g., Techniques for Locating Genes HindII, SmaI). ○ Historical Methods: Before 1970, locating genes Applications: Combining DNA from different organisms, was cumbersome and labor intensive. creating recombinant DNA. 5. Vectors : ○ Restriction Fragmentation: DNA molecules used to deliver genes into cells. Uses restriction enzymes to cut DNA into fragments. Types: Viral genomes, transposons, plasmids. ○ Compares fragments to map gene locations relative Features: Small, stable, identifiable markers, enable to each other. genetic expression. ○ Example: Complete gene map of the bacterium *H. 6. Gene Libraries : influenzae* in 1995. Collections of clones containing genetic material. ○ Applications: Simplifies isolating specific genes for ○ Fluorescent In Situ Hybridization (FISH): Uses fluorescent DNA probes to hybridize with target research. genes.  ○ Visualized under a fluorescent microscope to locate DNA Technology in Functional Genomics, Medicine, and specific genes and microbes. Agriculture ○ Applications: Disease diagnosis, identifying Functional Genomics microbes in environmental samples, analyzing ○ Gene Knockout: Removing a gene to observe biofilms. phenotypic changes. Genomics and Nucleotide Sequencing ○ E. coli Keio Knockout Collection: 3985 strains with single nonessential genes removed. ○ Genomics: Sequencing and analyzing the nucleotide bases of genomes. ○ Gene Overexpression: Enhancing transcription or translation to increase gene product abundance. ○ Early methods involved selective cleavage of DNA and mapping short molecules. Microbial Community Studies ○ Sanger Sequencing: Uses modified nucleotides to DNA Sequencing: Reveals genetic diversity of terminate DNA replication. uncultured microorganisms. ○ Automated sequencing with fluorescent dyes for Next Generation Sequencing: Rapid and cost effective each nucleotide. analysis of microbiomes. ○ Significant achievements include the Human Pharmaceutical and Therapeutic Applications Genome Project in 2001. ○ Recombinant DNA Technology: Synthesis of ○ Next Generation Sequencing (NGS): pharmaceuticals like insulin and interferon. Massively parallel sequencing, allowing hundreds of millions of DNA fragments to be sequenced simultaneously. ○ Vaccine Production: Safer subunit vaccines Four color reversible termination sequencing involves produced by inserting antigen genes into vectors. stopping DNA synthesis after each nucleotide addition, ○ Gene Therapy: Replacing missing or defective recording the sequence, and then removing the dye and stop genes with normal copies. group to continue. ○ Applications: Sequencing complete genomes of Agricultural Applications pathogens, developing drugs and therapies, relating ○ Transgenic Organisms: GMOs engineered for DNA sequences to protein functions. specific traits like herbicide tolerance and pest ○ Example: Studies on *Deinococcus radiodurans* for resistance. radiation resistance, psychrophiles for enzymes ○ Herbicide Tolerance: Glyphosate tolerant crops active at low temperatures. enable weed control without damaging crops. Applications and Impact ○ Pest Resistance: Crops producing insecticidal proteins from Bacillus thuringiensis. ○ Industrial and Environmental : Enhanced production processes, bioremediation, resource ○ Nutritional Improvement: Adding genes for desired traits like salt tolerance and increased extraction. nutritional value. ○ Medical : Development of pharmaceuticals, gene therapy, diagnostics. Safety and Ethical Concerns ○ Research : Simplified gene isolation, genetic ○ Debate: Controversy over long term effects and mapping, and sequencing. unforeseen problems. Summary of Tools and Techniques ○ Regulatory Measures: Implemented to prevent accidental release of altered organisms. ○ Mutagens : Induce genetic changes. ○ Reverse Transcriptase : Converts RNA to cDNA for ○ Ethical Issues: Privacy, ownership of genetically modified organisms, and forced genetic expression in prokaryotic cells. manipulations. ○ ynthetic Nucleic Acids : Created in vitro for various applications. Future Considerations ○ Restriction Enzymes : Cut DNA at specific sites for ○ Emerging Technologies: Continued advancement combining DNA fragments. in gene editing and its implications on society. ○ Vectors : Deliver genes into cells. ○ Ethical Considerations: Addressing societal ○ PCR : Amplifies specific DNA segments for analysis. concerns as genomic technologies evolve.