RDNA Technology can be used for the improvement of DNA

Vectors

1. Genetic Engineering 22BBT5CA LIGATION Unit I - Lecture 02 22BBT5CA - Genetic Engineering - Dr S Aravindh

2. What is DNA Ligation? Definition : DNA ligation is the process of joining two DNA strands together by forming a covalent bond between the phosphate backbone of the DNA. Enzyme : DNA ligase. Importance : - Recombinant DNA technology: Creating recombinant DNA molecules. - DNA repair: Essential for cellular DNA repair mechanisms. - Genetic engineering: Used in cloning, gene splicing, and synthetic biology. Unit I - Lecture 02 22BBT5CA - Genetic Engineering - Dr S Aravindh

3. Discovery of DNA Ligase Year: 1967 Researchers: Oliver H. Low and Jerard Hurwitz Significance: - Pioneering discovery in molecular biology. - Enabled advancements in genetic engineering and biotechnology. Unit I - Lecture 02 22BBT5CA - Genetic Engineering - Dr S Aravindh Jerard Hurwitz (1928-2019)

4. Types of DNA Ligases ATP-dependent DNA Ligases: - Found in eukaryotic cells. - Use ATP as a cofactor. NAD+-dependent DNA Ligases: - Found in bacteria (e.g., E. coli). - Use NAD+ as a cofactor. T4 DNA Ligase: - Derived from bacteriophage T4. - Most commonly used in laboratory settings. Unit I - Lecture 02 22BBT5CA - Genetic Engineering - Dr S Aravindh

5. Mechanism of Ligation Step 1: Enzyme Adenylylation : - Ligase enzyme forms a covalent complex with AMP. Step 2: Adenylyl Transfer to DNA: - Transfer of AMP to the 5’ phosphate of the DNA nick. Step 3: Nick Sealing: - Formation of a phosphodiester bond between adjacent nucleotides. Cofactors: - ATP or NAD+ depending on the type of ligase. Unit I - Lecture 02 22BBT5CA - Genetic Engineering - Dr S Aravindh

6. Unit I - Lecture 02 22BBT5CA - Genetic Engineering - Dr S Aravindh

7. Laboratory Ligation Common Enzyme: T4 DNA Ligase - Efficient at ligating sticky ends and blunt ends. Types of DNA Ends: Sticky Ends: - Overhanging ends created by restriction enzymes. - Facilitates directional cloning. Blunt Ends: - No overhangs; both ends are flush. - Requires higher ligase concentration for effective ligation. Unit I - Lecture 02 22BBT5CA - Genetic Engineering - Dr S Aravindh

8. Factors Affecting Ligation DNA Concentration: - Optimal concentration necessary for effective ligation. Ligase Concentration: - Higher concentration needed for blunt-end ligation. Temperature: - Typically performed at 16°C for optimal enzyme activity. Buffer Composition: - Buffer must contain appropriate ionic strength and cofactors (ATP or NAD+). Unit I - Lecture 02 22BBT5CA - Genetic Engineering - Dr S Aravindh

9. Effect of DNA Concentration High DNA Concentration: - Promotes intramolecular ligation (circularization). Low DNA Concentration: - Promotes intermolecular ligation (joining of separate fragments). Optimal Concentration: - Dependent on the type of ends (sticky vs. blunt). Unit I - Lecture 02 22BBT5CA - Genetic Engineering - Dr S Aravindh

10. Effect of Ligase Concentration Sticky-End Ligation: - Lower concentration sufficient due to complementary base pairing. Blunt-End Ligation: - Higher concentration needed due to lack of base pairing. - Addition of PEG (polyethylene glycol) can enhance ligation efficiency. Unit I - Lecture 02 22BBT5CA - Genetic Engineering - Dr S Aravindh

11. Temperature and Buffer Composition Temperature: - Optimal at 16°C for T4 DNA ligase. - Higher temperatures may denature the enzyme. Buffer Composition: - Includes ATP (for T4 DNA ligase) or NAD+ (for bacterial ligases). - Magnesium ions (Mg2+) as a cofactor. Unit I - Lecture 02 22BBT5CA - Genetic Engineering - Dr S Aravindh

12. Sticky-End Ligation Mechanism: - Overhanging ends of DNA fragments anneal through complementary base pairing. - T4 DNA ligase forms phosphodiester bonds. Directional Cloning: - Ensures the insert is ligated in the correct orientation. Dephosphorylation: - Prevents self-ligation of the vector. Unit I - Lecture 02 22BBT5CA - Genetic Engineering - Dr S Aravindh

13. Blunt-End Ligation Mechanism: - Blunt ends directly ligated without base pairing. - Requires higher ligase concentration. PCR Products: - Often have blunt ends. - Phosphorylation of PCR products with T4 polynucleotide kinase. Efficiency: - Generally lower than sticky-end ligation. - Enhanced by increasing enzyme concentration and using PEG. Unit I - Lecture 02 22BBT5CA - Genetic Engineering - Dr S Aravindh

14. General Guidelines for Cloning DNA Concentration: - Use 1:3 molar ratio of vector to insert. Ligase Amount: - Use 1 unit of ligase for sticky ends; higher for blunt ends. Incubation Time and Temperature: - 16°C overnight for optimal results. Troubleshooting: - Ensure DNA ends are compatible. - Check for contamination or degraded DNA. Unit I - Lecture 02 22BBT5CA - Genetic Engineering - Dr S Aravindh

15. Advanced Ligation Methods Topoisomerase-Mediated Ligation: - Faster and more efficient. - Used in commercial cloning kits (e.g., TOPO cloning). Homologous Recombination: - Used in yeast and bacteria. - Enables precise insertion and replacement. Unit I - Lecture 02 22BBT5CA - Genetic Engineering - Dr S Aravindh

16. Applications of DNA Ligases Molecular Cloning: - Creation of recombinant DNA molecules. - Gene cloning and expression. DNA Repair: - Essential in cellular DNA repair pathways. - Fixes nicks and breaks in DNA. Genetic Engineering: - Creation of genetically modified organisms (GMOs). - Synthetic biology applications. Unit I - Lecture 02 22BBT5CA - Genetic Engineering - Dr S Aravindh

17. Conclusion Summary of Key Points: - DNA ligation is crucial for molecular biology. - Involves joining DNA fragments via ligase enzymes. - Various factors affect ligation efficiency. Applications in cloning, DNA repair, and genetic engineering. Future Prospects: - Advances in ligation techniques. - Broader applications in synthetic biology and medicine. Unit I - Lecture 02 22BBT5CA - Genetic Engineering - Dr S Aravindh