Recombinant dna technology

in this topic we will discuss the following contents
Introduction.
Cloning.
Discovery.
Molecules need in rDNA technology.
Enzymes.
Vectors.
Procedure or steps involves in rDNA technology.
Application of rDNA technology.
Advantages and disadvantages.

1. RECOMBINANT DNA TECHNOLOGY Muhammad K amil Khan 1 st S emister M.phil Microbiology 1

2. Contents Introduction. Cloning. Discovery. Molecules need in rDNA technology. Enzymes. Vectors. Procedure or steps involves in rDNA technology. Application of rDNA technology. Advantages and disadvantages. 2

3. Introduction The technique through which we join the DNA molecules together from two different species in lab. Then these joining DNA molecules are inserted into a host organism to produce new genetic combinations that are of valuable to science, medicine, agriculture, and industry. 3

4. Cloning In recombinant DNA technology we usually perform the cloning process. In biotechnology cloning is refers to the process of creating clones of organisms or copies of cells or DNA fragments. In cloning we usually clone the DNA or genes. 4

5. Discovery Paul Berg generated rDNA technology in 1972 Cohen & Boyer in 1973 produced first plasmid vector capable of being replicated within a bacterial host 5

6. Molecules need in cloning Target DNA having genes of interest Enzymes Vectors Host cell i.e E. coli 6

7. Enzymes used for rDNA technology DNA ligase • Bind to DNA molecules. Type II restriction endonuclease • Cleaves DNA at specific sites. Polynycleotide Kinase • Adds a phosephate to the 5'-OH end of a polynucleotide. Terminal transferase • Adds homopolymer tails to the 3'-OH ends. Alkaline phosphatase • Removes terminal phosphates. Polymerase enzymes that add nucleotides. 7

8. Vectors Vectors are the vehicles or delivery system that can deliver or transfer our target DNA having gene of interest in to the host cell. Types of vectors Expression vectors. Cloning vectors. Plasmid. Cosmid . Phage vector. Bacterial artificial chromosomes (BAC). Yeast artificial chromosomes (YAC). 8

9. Procedure or steps need in rDNA Isolating of DNA. Cutting of DNA. Joining of DNA. Transferring of the rDNA in to host. Amplification of DNA. Screening. 9

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11. 1. Isolation 11

12. 2. Cutting of DNA molecules The same type 2 R.E are used to cut DNA molecules having gene of interest as well as vectors i.e EcoR1 or BamH1 etc restriction endonuclease to produce sticky ends 12

13. 3. Joining of DNA molecules Once the target DNA is uptake by the vectors then the gaps is still remaining which is sealed by ligase enzymes Finally the vector and target DNA are joined and we called that rDNA vector 13

14. 4. Transferring of the rDNA into the host The recombinant dna vector should be transfer into the host cell i.e E.coli It can be possible by making the cell competent Treat the host cell by cacl2 or Mgcl2. Electric shock to host cell. 14

15. 5. Amplification of rDNA vector Once the host cell uptake the rDNA vector then we allow the cell inside the media to multiply and divided Thus our target DNA is also replicated because transcription, translation and replication is always going on inside host cell and we get cDNA or gene library Finally the target amplified. 15

16. 6. Screening Now to identify that which cell have our rDNA vector we perform screening technique. It may be Blue white screening. Using selectable marker genes. 16

17. Application of rDNA technology Agriculture: growing crops of your choice (GM food),pesticide resistant crops, fruits with attractive colors, all being grown in artificial conditions. Pharmacology: artificial insulin production, drug delivery to target sites. Medicine: gene therapy, antiviral therapy, vaccination, synthesizing clotting factors. 17

18. Advantages To isolate and characterize a gene. • To make desired alterations in one or more isolated genes. • To return altered genes to living cells. • Artificially synthesize new gene. • Understanding the hereditary diseases and their cure • Improving human genome. Improve fertilization inside woman 18

19. Disadvantages Expensive method Sometime the vector self anneal with each other Difficult process Effect on health Can take long time Can cause mutation 19

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