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Biotechnology: Restriction Enzyme and Recombinant DNA Technology
- 1. Biotechnology: Restriction Enzyme and Recombinant DNA Technology Pooja Sarda Overview Biotechnology Restriction Enzyme Recombinant DNA Technology Separation and isolation of DNA fragments Thank You Biotechnology: Restriction Enzyme and Recombinant DNA Technology Pooja Sarda Sep 24,2020
- 2. Biotechnology: Restriction Enzyme and Recombinant DNA Technology Pooja Sarda Overview Biotechnology Restriction Enzyme Recombinant DNA Technology Separation and isolation of DNA fragments Thank You Overview What is Biotechnology? Restriction Enzyme Recombinant DNA Technology Separation and Isolation of DNA Fragment
- 3. Biotechnology: Restriction Enzyme and Recombinant DNA Technology Pooja Sarda Overview Biotechnology Restriction Enzyme Recombinant DNA Technology Separation and isolation of DNA fragments Thank You Biotechnology Technology utilizing living organism or biological system of organism Developing modified products Technological application that using biological systems, living or- ganism, or derivatives to make or modify products or processes for specific use Principles of Biotechnology Genetic Engineering: Introduction of genetic material (DNA/RNA) into host cell altering its phenotype. Bioprocess Engineering: Manufacturing products by chemical engi- neering processes by maintaining aseptic environment (antibiotics, vaccines, enzymes etc.)
- 4. Biotechnology: Restriction Enzyme and Recombinant DNA Technology Pooja Sarda Overview Biotechnology Restriction Enzyme Recombinant DNA Technology Separation and isolation of DNA fragments Thank You Restriction Enzyme 1963: 2 Enzymes are responsible for restricting the growth of bac- teriophage in Escherichia coli were isolated First endonuclease found was Hind II Types: Restriction endonuclease and restriction exonuclease Naming of Restriction enzymes (Eco RI) Eco: Escherichia coli (Name of the bacteria) R: RY strain I: Roman number indicating the order in which enzyme was isolated More than 900 restriction enzymes that have been isolated from over 230 bacterial strains. RE recognize and cleave DNA within sequences of 4-8 ntds. se- quences are often referred to as palindromes. Axis of Symmetry /- cutting site; *- methylated site
- 5. Biotechnology: Restriction Enzyme and Recombinant DNA Technology Pooja Sarda Overview Biotechnology Restriction Enzyme Recombinant DNA Technology Separation and isolation of DNA fragments Thank You Restriction Enzyme Restriction endonuclease functions by ‘inspecting’ the length of a DNA sequence. Once it finds its specific recognition sequence, it will bind to the DNA and cut each of the two strands of the double helix at specific points in their sugar -phosphate backbones. Each restriction endonuclease recognises a specific palindromic nu- cleotide sequences in the DNA. Figure: Steps in formation of recombinant DNA by action of restriction endonuclease enzyme EcoRI Figure: Restriction enzymes and their recognition sites
- 6. Biotechnology: Restriction Enzyme and Recombinant DNA Technology Pooja Sarda Overview Biotechnology Restriction Enzyme Recombinant DNA Technology Separation and isolation of DNA fragments Thank You Recombinant DNA Technology Restriction endonucle- ases are used in genetic engineering to form re- combinant molecules of DNA, which are composed of DNA from different sources/genomes. When cut by the same re- striction enzyme, the resul- tant DNA fragments have the same kind of sticky- ends and these can be joined together (end-to- end) using DNA ligases Figure: Diagrammatic representation of Recombinant DNA Technology
- 7. Biotechnology: Restriction Enzyme and Recombinant DNA Technology Pooja Sarda Overview Biotechnology Restriction Enzyme Recombinant DNA Technology Separation and isolation of DNA fragments Thank You Separation and isolation of DNA fragments Gel electrophoresis Most commonly used matrix for gel electrophoresis is agarose which is a natural polymer extracted from sea weeds.. DNA fragments are negatively charged molecules they can be separated by forcing them to move towards the anode under an electric field through a medium/matrix. The DNA fragments separate (resolve) according to their size through sieving effect provided by the agarose gel. DNA is Visualized by Ethidium Bromide. EtBr emits Orange red color under Uv light- Gel Documentation system or transillumina- tor. Cutting the bands. Elution of DNA. Figure: Agarose gel electrophoresis of DNA migration (Lane 1-undigested DNA, Lane 2,3,4- digested DNA)
- 8. Biotechnology: Restriction Enzyme and Recombinant DNA Technology Pooja Sarda Overview Biotechnology Restriction Enzyme Recombinant DNA Technology Separation and isolation of DNA fragments Thank You Thank you!