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lecture 1 introduction to Recombinant DNA technology

RECOMBINANT DNA TECHNOLOGY<br>DNA technology<br>DNA technology is a term that describes the collective techniques for obtaining, amplifying, and manipulating speciufb01c DNA fragments. Since the mid-1970s, the development of DNA technology has revolutionized the study of biology, opening many areas of research to molecular investigation.<br>Biotechnology: Technology involved in synthesis of artificial genes, repair of genes, product of recombinant DNA and manipulating them for the improvement in human beings, plants, animals and microbes is called biotechnology. The term was coined by Karl Ereky (1917).<br>Ge

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lecture 1 introduction to Recombinant DNA technology

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  1. Lecture No. 1 • Introduction to Recombinant DNA technology • DNA technology • Biotechnology • Recombinant DNA technology • Basic techniques • Steps involved Rab Nawaz Assistant prof. GPGC Dargai, Malakand

  2. RECOMBINANT DNA TECHNOLOGY • DNA technology • Definition: Collective techniques for obtaining, amplifying, and manipulating specific DNA fragments is called DNA technology. Rab Nawaz Assistant prof. GPGC Dargai, Malakand

  3. RECOMBINANT DNA TECHNOLOGY • Biotechnology: • Definition: Technology involved in synthesis of artificial genes, repair of genes, product of recombinant DNA and manipulating them for the improvement in human beings, plants, animals and microbes is called biotechnology. • The term was coined by Karl Ereky (1917). Rab Nawaz Assistant prof. GPGC Dargai, Malakand

  4. RECOMBINANT DNA TECHNOLOGY • Genetic Engineering: • Definition: The technique which alters the chemistry of genetic material (DNA or RNA) to introduce these into host organism is called genetic engineering. • Paul Berg is the father of genetic engineering (Noble Prize, 1980) as he was the first one to introduce a gene of SV-40 into a bacterium with the help of lambda phage. Rab Nawaz Assistant prof. GPGC Dargai, Malakand

  5. RECOMBINANT DNA TECHNOLOGY • Recombinant DNA technology: • Definition: DNA technology of producing Recombinant DNA is termed as Recombinant DNA technology. • Recombinant DNA refers to the creation of new combinations of DNA segments that are not found together in nature. • Gene of interest (i.e insulin gene) cut and then inserted in to a vector (plasmid) the resulted DNA is Recombinant DNA. • Recombinant DNA technology began in 1971 Rab Nawaz Assistant prof. GPGC Dargai, Malakand

  6. RECOMBINANT DNA TECHNOLOGY Rab Nawaz Assistant prof. GPGC Dargai, Malakand

  7. Steps Involved In Recombinant DNA technology • Selection and isolation of the genetic material (DNA). • Cutting of DNA at specific location (restriction enzymes). • Joining DNA • Vectors/ Carrier • Transformation • Growth of transformed organism. • Obtaining foreign gene product. • Downstream processing Rab Nawaz Assistant prof. GPGC Dargai, Malakand

  8. Selection and isolation of the genetic material (DNA). • Genomic DNA:This DNA is obtained directly from the chromosomes of the organism under study. It is the most straightforward source of DNA. It needs to be cut up before cloning is possible. • Complementary DNA (cDNA):The cDNA is made from mRNA with the use of a special enzyme called reverse transcriptase. . cDNA does not need to be cut in order to be cloned. • Chemically synthesized DNA: . If the DNA sequence is known (often from a complete genome sequence), then the gene can be synthesized chemically by using automated techniques. Rab Nawaz Assistant prof. GPGC Dargai, Malakand

  9. 2) Cutting of DNA at specific location (restriction enzymes). • Restriction enzymes are the scissors of molecular genetics. • Restriction enzymes (RE) are endonucleases that will recognize specific nucleotide sequences in the DNA and break the DNA chain at those points. • A variety of RE have been isolated and are commercially available • Most cut at specific palindromic sites in the DNA (sequence that is the same on both antiparallel DNA strands). • These cuts generates “sticky or overhanging ends” or a blunt ends. Rab Nawaz Assistant prof. GPGC Dargai, Malakand

  10. 2) Cutting of DNA at specific location (restriction enzymes). Rab Nawaz Assistant prof. GPGC Dargai, Malakand

  11. 2) Cutting of DNA at specific location (restriction enzymes). Rab Nawaz Assistant prof. GPGC Dargai, Malakand

  12. 2) Cutting of DNA at specific location (restriction enzymes). Rab Nawaz Assistant prof. GPGC Dargai, Malakand

  13. 3) JOINING DNA • The DNAs are mixed together in a tube. • If both have been cut with the same RE, the ends will match up because they are sticky. DNA ligase is the glue of molecular genetics that holds the ends of the DNAs together. • DNA ligase creates a phosophodiester bond between two DNA ends Rab Nawaz Assistant prof. GPGC Dargai, Malakand

  14. 3) JOINING DNA Rab Nawaz Assistant prof. GPGC Dargai, Malakand

  15. 4)VECTOR or Carrier • Vector in genetic engineering is usually a DNA segment used as a carrier for transferring selected DNA into living cells. • Requirements for a cloning vector • Should be capable of replicating in host cell • Should have convenient RE sites for inserting DNA of interest • Should have a selectable marker to indicate which host cells received recombinant DNA molecule • Should be small and easy to isolate Rab Nawaz Assistant prof. GPGC Dargai, Malakand

  16. 4)VECTOR or Carrier • Some of the vectors are: • Plasmid : Plasmids are extrachromosomal, closed circular double stranded molecules of DNA present in most prokaryotes. pBR 322 was one of the first widely used cloning vectors, which contains both ampR and tetR genes. • Phage : It is constructed from phage ë and acts as bacteriophage cloning vector. • Cosmid : Hybrid between plasmid and phage chromosome give rise to cosmidvectors. • Besides all these there are artificial chromosomes like a) BAC : Bacterial artificial chromosome b) YAC : Yeast artificial chromosome c) MAC : Mammalian artificial chromosome Rab Nawaz Assistant prof. GPGC Dargai, Malakand

  17. 4)VECTOR or Carrier Rab Nawaz Assistant prof. GPGC Dargai, Malakand

  18. 5)Transformation • To recover large amounts of the recombinant DNA molecule, it must be amplified. • The transfer of recombinant DNA into host cell for multiplication is called transformation. • Transformation involve the following methods • Heat shock method (plasmid DNA to cells chilled on ice or heat the DNA the cell mixture) • Electroporation (brief pulse of high voltage electricity to create tiny holes in the bacteria cell wall that allow the DNA to enter) • Viruses • Microinjection • Gene gun method Rab Nawaz Assistant prof. GPGC Dargai, Malakand

  19. 6) Growth of transformed organism • When the cell divides, the replicated recombinant molecules go to both daughter cells which themselves will divide later. Thus, the DNA is amplified. Rab Nawaz Assistant prof. GPGC Dargai, Malakand

  20. Rab Nawaz Assistant prof. GPGC Dargai, Malakand

  21. Rab Nawaz Assistant prof. GPGC Dargai, Malakand

  22. Thank you

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