A Complete Guide to Gene Cloning: From Basic to Advanced

This comprehensive guide to gene cloning provides beginning and advanced readers with the background, standard techniques, practical applications, and ethical and safety considerations in the field. A one-stop reference for students, researchers in academia and industry, and anyone interested in a thorough but accessible overview.

Auteur

Nayana Patil, PhDAssociate Professor, School of Bioengineering Sciences and Research, MIT ADT University Aruna Sivaram, PhDAssociate Professor, School of Bioengineering Sciences and Research, MIT ADT University

Contenu

1. The recombinant DNA technology era 1.1 Important timelines 1.2 Scientist and their contribution to recombinant DNA technology 1.3 Recent advances 2. Vectors: Guide to gene delivery vehicles 2.1 Plasmid, Cosmid, Phasmid, Bacteriophage 2.2 YAC and BAC 2.3 Shuttle vector 2.4 Expression vector 3. Gene isolation methods: Beginners guide 3.1 Isolation of genomic and PCR amplification 3.2 Gene synthesis 3.3 cDNA library 3.4 Genomic library 4. Gene cloning strategies: overview 4.1 Restriction and ligation-based cloning 4.2 Ligation-independent cloning 4.3 PCR-based cloning4.4 Recombination-based cloning 4.5 Mating-Assisted Genetically Integrated Cloning 5. DNA cutters and connecters in recombinant DNA technology 5.1 RM system 5.2 Restriction enzymes 5.3 Sequence specific, G+C content, Methylation 5.4 Elimination of RM in host 6 Fundamental techniques of recombinant DNA transfer 6.1 Chemical transformation in Bacteria, Fungi and animal cell 6.2 Physical transformation in Bacteria, Fungi and animal cell 7. Screening of recombinants 7.1 Colony hybridisation 7.2 Antibiotic based screening 7.3 Colony PCR 7.4 Plasmid Miniprep 7.5 Southern blotting 7.6 DNA Sequencing 7.7 Protein expression SDS PAGE, western blotting 8. Recent Trends and Advances 8.1 Basics of gene editing 8.2 Zinc finger nucleases 8.3 Transcription activators like effectors 8.4 RNA interference 8.4 CRISPR Cas system 8.5 Cre-lox mechanism 9. Application of Recombinant DNA Technology 9.1 Health, Medical and Diagnostics, Pharmaceuticals, Agriculture, , Industry 9.2 Health: Gene therapy, Diagnostics, 9.3 Pharmaceuticals: Vaccines, Insulin, Antibiotics, antibodies 9.4 Environment: Bioremediation, Bio indicators 9.5 Industry: Enzyme 9.6 Agriculture: Yield improvement, tolerant plants, transgenic for secondary metabolites 10. Ethical and safety concerns of recombinant DNA technology 10.1 Questionable knowledge dissemination 10.2 Uncertainties about misuse of technology 10.3 Potential risk of engineered organisms