RECOMBINANT DNA TECHNOLOGY PRESENTATIONS

rDNA Technology

1. PADMASHREE DR.D.Y.PATIL COLLEGE OF PHARMACY AKURDI TOPIC NAME- RECOMBINANT DNA TECHNOLOGY SUBJECT NAME – CELLULAR AND MOLECULAR PHARMACOLOGY GUIDED BY- DR. PAWAN WANKHADE SIR PRESENTED BY – DHANASHRI PRAKASH SONAVANE

2. INTRODUCTION: Recombinant DNA technology Recombinant DNA technology  involves using enzymes and various laboratory techniques to manipulate and isolate DNA segments of interest . This method can be used to combine (or splice) DNA from different species or to create genes with new functions. The resulting copies are often referred to as recombinant DNA. Ex: DNA Comprising animal gene may be recombined with DNA From Bacteria.

3. Principle Every protein is encoded by a certain gene. If the gene can be identified, then that can be used to produce a large quantity of the same gene or product encoded by that gene. The gene of interest can be transferred to a vector DNA to produce recombinant DNA or cloned gene. Vectors are self-replicating inside an appropriate host cell. Introduction of recombinant vectors into host cells cause multiplication of the cloned gene. Expression of the cloned gene produces desired protein.

4. Discovery Of Recombinant DNA Technology Discovery of DNA Structure-Watson and Crick 1953. Isolation of DNA Ligase in 1967. rDNA Technology Developed by Paul Berg. Cohen and Bayer in 1973 produced 1st plasmid vector capable of being replicated with bacterial host.

5. GOALS OF rDNA TECHNOLOGY To isolate and characterize gene. To make desired alteration in one or more isolated gene. To return altered gene to living cell. Artificially synthesize new gene. Alteration the genome of an organization. Understand heredity disease and their cure. Improve Human Genome.

6. Step of rDNA Technology Isolation of desired DNA. Isolation of vector. Chimeric DNA production. Introduction of the recombinant DNA into host cell. Multiplication and selection of cell with DNA Expression of gene -Product. Separation and purification of the product.

7. Isolation of Desired piece of DNA which get separated by electrophoresis & identified by southern blotting technique.

8. 2. Isolation of vectors:

9. 3.Production/Hybridization of (Chimeric) Base DNA

10. 4 . Introduction of the recombinant DNA into host cell:

11. 5 . Multiplication of Host cell to selection of cell containing Chimeric DNA esters Done by Hybridization technique with the help of Probe Antibiotic Br by using resistance. Expression of gene to produce desired product: After this desired piece of gene produce desired protein Process on transcription & translation. 7. Separation and purification of the product.

12. Study various tools used in rDNA technology Enzymes Exonuclease-cut outer nucleotide. Endonuclease - cut act on internal Phosphodiesterase linkage. Restriction endonuclease (also called as molecule seizure). Restriction endonuclease 1 Restriction endonuclease2 Restriction endonuclease3  1 and 2 required ATP they cut the DNA molecules away from recognition sequence.  but 2 is important because it cut the DNA molecule within the sequence.

13. recognition sequence is 4-6 nucleotides and it 13 plandromic (rate similar in either direction. EX: --G A A T T C-- --C T T A A G-- Single Strand of E.Coli Bacteria They act on ; --G A A T T C-- Then You Will Get --C T T A A G-- --C T T A A G-- -- G A A T T C-- Separated by Sticky And Blunt End.

14. B. DNA Ligase: (DNA joining enzyme are also called molecular glue) require ATP & Phosphodiesterase linkage Bond formation. C. Polymerase D. Alkaline Phosphate E. Reverse Transcriptase

15. Vectors Vector is a DNA molecule in to which a gene is inserted to construct recombinant DNA molecule and it is also capable of  replicate in a host organism.  It is also otherwise known as vehicle because the vector transports the inserted gene to host organisms.

16. Gene Transfer Technique Transformation Transduction Electroporation Conjugation Direct Transfer of DNA Lipofection

17. Electroporation: It is based on the principle that high voltage electric pulses can induce cell plasma membranes to fuse. It is a technique involving electric field-mediated membrane permeation. Electric shock can also induce cellular uptake of exogenous DNA (believed to be pores formed by electric pulses) from the suspending solution. Conjugation: In this method, two live bacteria (a donor and a recipient) come together, join by cytoplasmic bridges and transfer single stranded DNA from donor to recipient. Inside the recipient cell, the new DNA may integrate with the chromosome (rare) or may remain free (as is the case with Plasmids).

18. Transduction: Sometimes the foreign DNA can be packed inside animal viruses. These viruses can naturally infect the cells and introduce the DNA into host cells. The transfer of DNA by this approach is referred to as transduction. Direct Transfer of DNA : Microinjection and particle bombardment are the two techniques commonly used to directly transfer the DNA into the cell nucleus. Lipofection: The liposome mediated gene transfer is referred to as lipofection. Liposomes are circular lipid molecules, which have an aqueous interior that can carry nucleic acids.

19. Transformation It is the method of introducing foreign DNA in bacteria cells ex: E.Coli The uptake of plasmid DNA by E.Coli is carried out in: Ice-cold CaCl ₂ (0-5°C). Ice-cold CaCl ₂ affects the cell wall, breaks at localised regions and is also responsible for binding of DNA to cell surface. A subsequent heat shock (37°C-45°C for about 90 seconds). A brief heat shock (e.g. the sudden increase in temperature from 5°C to 40°C) stimulates DNA uptake.