rDNA Technology

rDNA technology , what it is , how it works , components , will be shown in this slide.

1. Rdna tECHNOLOGY pRESENted by: Srishti patel

2. Index S. No. Topic Slide No. 1. Definition 3 2. History 4 3. 3 Tools of Recombinant DNA Technology 5-8 4. 7 Steps of Recombinant DNA Technology 9-17 5. Application of Recombinant DNA Technology 18 6. Limitations of Recombinant DNA Technology 19 7. Reference 20

3. DefinitioN A series of procedures that are used to join together (recombine) DNA segments. A recombinant DNA molecule is constructed from segments of two or more different DNA molecules. Under certain conditions, a recombinant DNA molecule can enter a cell and replicate there, either on its own or after it has been integrated into a chromosome.

4. HIStory The possibility for recombinant DNA technology emerged with the discovery of  restriction enzymes  in 1968 by Swiss microbiologist  Werner Arber 00, Hamilton O. Smith, and Daniel Nathans..  American microbiologist  Hamilton O. Smith  purified type II restriction enzymes, which were found to be essential to genetic engineering for their ability to cleave at a specific site within the DNA. American molecular biologist  Daniel Nathans  helped advance the technique of DNA recombination in 1970–71 and demonstrated that type II enzymes could be useful in genetic studies.  About the same time, American biochemist  Paul Berg developed methods for splitting DNA molecules at selected sites and attaching segments of the molecule to the DNA of a virus or  plasmid , which could then enter bacterial or animal cells . In 1973 American biochemists  Stanley N. Cohen  and Herbert W. Boyer became the first to insert recombined genes into bacterial cells, which then reproduced.

5. 3 tool of recombinant dna technology

6. Restriction Enzymes These are enzymes which have restriction endonucleases that help in cutting, polymerases that aid in synthesis of DNA and ligases that facilitate binding. The restriction endonucleases used in recombinant DNA technology have a vital role in outlining the location at which the desired gene of interest is introduced into the vector genome. These are of two types usually, named as endonucleases and exonucleases. The endonucleases cut within the strand of DNA whereas the exo nucleotides are involved in cutting the ends of the DNA. The restriction endonucleases are specific to palindromic sequences and cut the DNA at specific points. There are 3 chief types of restriction endonuclease enzymes: Type-I Restriction Endonucleases, Type-II Restriction Endonucleases and Type-III Restriction Endonucleases. They inspect the length of DNA and cut at the specific site known as the restriction site. This creates sticky ends in the sequence. The gene of interest and the vectors are cut by the same restriction enzymes to acquire the corresponding sticky ends, after which ligases help in binding the sticky ends.

7. Vectors The vectors help in carrying and integrating the desired gene. These form a very important part of the tools of recombinant DNA technology as they are the ultimate vehicles that carry forward the desired gene into the host organism. Plasmids and bacteriophages are the most common vectors in recombinant DNA technology that are used as they have a very high copy number. The vectors are made up of an origin of replication- This is a sequence of nucleotide from where the replication starts, a selectable marker – constitute genes which show resistance to certain antibiotics like ampicillin; and cloning sites – the sites recognized by the restriction enzymes where desired DNAs are inserted.

8. Host Organism The organism in which the recombinant DNA is introduced is called the host organism. It is the ultimate tool into which the vector drives the gene of interest using the enzymes. Techniques like microinjection, gene gun, biolistic are employed to insert the recombinant DNA within the organism. This can be also carried out using alternate heating and cooling or use of calcium ions.

9. 7 Steps of Recombinant dna Technology

10. Isolation of Genetic Material The first step in rDNA technology is to isolate the desired DNA in its pure form i.e. free from other macromolecules. Since DNA exists within the cell membrane along with other macromolecules such as RNA, polysaccharides, proteins, and lipids, it must be separated and purified which involves enzymes such as lysozymes, cellulase, chitinase, ribonuclease, proteases etc. Other macromolecules are removable with other enzymes or treatments. Ultimately, the addition of ethanol causes the DNA to precipitate out as fine threads. This is then spooled out to give purified DNA.

11. Restriction Enzyme Digestion Restriction enzymes act as molecular scissors that cut DNA at specific locations. These reactions are called ‘restriction enzyme digestions’. They involve the incubation of the purified DNA with the selected restriction enzyme, at conditions optimal for that specific enzyme. The technique ‘Agarose Gel Electrophoresis’ reveals the progress of the restriction enzyme digestion. This technique involves running out the DNA on an agarose gel. On the application of current, the negatively charged DNA travels to the positive electrode and is separated out based on size. This allows separating and cutting out the digested DNA fragments. The vector DNA is also processed using the same procedure.

12. Amplification Using PCR Polymerase Chain Reaction or PCR is a method of making multiple copies of a DNA sequence using the enzyme – DNA polymerase in vitro. It helps to amplify a single copy or a few copies of DNA into thousands to millions of copies. PCR reactions are run on ‘thermal cyclers’ using the following components: Template – DNA to be amplified Primers – small, chemically synthesized oligonucleotides that are complementary to a region of the DNA. Enzyme – DNA polymerase Nucleotides – needed to extend the primers by the enzyme. The cut fragments of DNA can be amplified using PCR and then ligated with the cut vector.

13. Ligation of DNA Molecules

14. Ligation of DNA Molecules The purified DNA and the vector of interest are cut with the same restriction enzyme. This gives us the cut fragment of DNA and the cut vector, that is now open. The process of joining these two pieces together using the enzyme ‘DNA ligase’ is ‘ligation’. The result­ing DNA molecule is a hybrid of two DNA molecules – the interest molecule and the vector. In the ter­minology of genetics this intermixing of dif­ferent DNA strands is called recombination. Hence, this new hybrid DNA molecule is also called a recombinant DNA molecule and the technology is referred to as the recom­binant DNA technology.

15. Insertion of Recombinant DNA Into Host In this step, the recombinant DNA is introduced into a recipient host cell mostly, a bacterial cell. This process is ‘Transformation’. Bacterial cells do not accept foreign DNA easily. Therefore, they are treated to make them ‘competent’ to accept new DNA. The processes used may be thermal shock, Ca++ ion treatment, electroporation etc.

16. Obtaining Foreign Gene Product The recombinant DNA multiplies in the host and is expressed as a protein, under optimal conditions. This is now a recombinant protein. Small volumes of cell cultures will not yield a large amount of recombinant protein. Therefore, large-scale production is necessary to generate products that benefit humans. For this purpose, vessels called bioreactors are used. Bioreactors are large containers with a continuous culture system, where the fresh medium is added from one side and used medium is taken out from another side.

17. Downstream Processing Before the protein is marketed as a final product, it is subjected to downstream processing which includes: Separation and purification. Formulation with suitable preservatives. Clinical trials to test the efficacy and safety of the product. Quality control tests.

18. Application of Recombinant DNA technology

19. Limitations of Recombinant DNA technology

20. Reference Britannica - https://www.britannica.com/science/recombinant-DNA-technology Wikipedia - https://en.wikipedia.org/wiki/Molecular_cloning Verma, P. S., & Agrawal, V. K. (2006). Cell Biology, Genetics, Molecular Biology, Evolution & Ecology (1 ed.). S .Chand and company Ltd. Klug, W. S., & Cummings, M. R. (2003). Concepts of genetics. Upper Saddle River, N.J: Prentice Hall. BYJU’S - https://byjus.com/biology/recombinant-dna-technology

21. Thank you! pRESENted by: Srishti patel