Topic – DNA Technology - PowerPoint Presentation

1. Topic –DNA TechnologyPresented byMs. P. H. GiriDepartment of MicrobiologyDeogiri College, Aurangabad

2. B.Sc T. Y. Semester VIPaper No. XIXRecombinant DNA TechnologyMs. Priyanka H. Giri

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5. Steps in RDT:

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16. Objectives of G.E.:To improve existing microbial processes through improving the existing cultures and discarding the unwanted by-products.Provide much benefit to branches of industrial microbiology, environmental microbiology, agricultural microbiology etc.Certainly genetically engineered micro-organisms such as bacteria and fungi have influenced the traditional processes of baking and cheese making.Much impact have been seen in production of life saving drugs and removing non functional genes and delivering the functional genes in animals.Several vaccines have been prepared and tested for its applicability.

17. Tools used for Gene cloning:Desired geneSuitable vectorDNA manipulating enzymes eg. RE and DNA ligase enzymesHost cell

18. DNA manipulating enzymes:The enzymes used in the RDT fall into four broad categories:1. Template dependent DNA polymerase2. Nucleases3. End modification enzymes4. Ligases

19. 1. Template dependent DNA polymeraseDNA polymerase enzyme that synthesize new poly-nucleotides complementary to an existing DNA or RNA template are included in this category.Different types of DNA polymerase used in gene manipulation are1. DNA polymerase I II. Reverse transcriptaseIII. Taq DNA polymerase

20. 1. DNA polymerase I (Kornberg Enzyme)It has both 3’-5’ and 5’-3’exonuclease activity and 5’-3’ polymerase activity.It is an enzyme that participates in prokaryotic DNA replication.Discovered by Arthur Kornberg in1956, It was the first known DNA polymerase.

21. II. Reverse transcriptaseAlso known as RNA dependent DNA polymerase. Working independently, Baltimore and Temin discovered reverse transcriptase , an enzyme that synthesizes DNA from RNA.H. Temin and D. Baltimore showed that the retroviruses namely Rous sarcoma virus and the AIDS virus can make an RNA-dependent DNA polymerase that synthesizes a DNA copy (cDNA) of viral RNA. The cDNA is made into double stranded molecule which is integrated into host genome where it is replicated and transcribed with host DNA; the topsy curvy path of flow of genetic information, i.e., RNA-DNA-RNA-protein. The enzyme was termed as reverse transcriptase.

22. It is very useful in constructing cDNA library.cDNA is a complementary DNA. It is produced using mRNA by the process called reverse transcription.Mostly for eukaryotic organisms cDNA library is constructed. WHY?

23. III. Taq DNA polymeraseDerived from thermostable bacterium, Thermus aquaticus. It operates at 72˚C and is reasonably stable above 90˚C and is used in PCR.

24. 2. NucleasesThese are enzymes which degrade DNA molecules by breaking the phosphodiester bonds that link one nucleotide to the next.There are two different kinds of nucleases:1. Exonuclease2. Endonuclease

25. Restriction Endonuclease:The term restriction enzyme originated from the studies of phage λ, a virus that infects bacteria, and the phenomenon of host-controlled restriction and modification of such bacterial phage or bacteriophage. The phenomenon was first identified in work done in the laboratories of Salvador Luria, Weigle and Giuseppe Bertani in the early 1950s.It was found that, for a bacteriophage λ that can grow well in one strain of Escherichia coli, for example E. coli C, when grown in another strain, for example E. coli K, its yields can drop significantly, by as much as 3-5 orders of magnitude

26. The host cell, in this example E. coli K, is known as the restricting host and appears to have the ability to reduce the biological activity of the phage λ. If a phage becomes established in one strain, the ability of that phage to grow also becomes restricted in other strains. In the 1960s, it was shown in work done in the laboratories of Werner Arber and Matthew Meselson that the restriction is caused by an enzymatic cleavage of the phage DNA, and the enzyme involved was therefore termed a restriction enzyme

27. The restriction enzymes studied by Arber and Meselson were type I restriction enzymes, which cleave DNA randomly away from the recognition site.In 1970, Hamilton O. Smith, Thomas Kelly and Kent Wilcox isolated and characterized the first type II restriction enzyme, HindII, from the bacterium Haemophilus influenzae.Restriction enzymes of this type are more useful for laboratory work as they cleave DNA at the site of their recognition sequence and are the most commonly used as a molecular biology tool.

28. Later, Daniel Nathans and Kathleen Danna showed that cleavage of simian virus 40 (SV40) DNA by restriction enzymes yields specific fragments that can be separated using polyacrylamide gel electrophoresis, thus showing that restriction enzymes can also be used for mapping DNA.For their work in the discovery and characterization of restriction enzymes, the 1978 Nobel Prize for Physiology or Medicine was awarded to Werner Arber, Daniel Nathans, and Hamilton O. Smith.

29. RE is a bacterial enzyme that cuts dsDNA by cleaving the phosphodiester bonds at specific sites knows as restriction sites.Recently more than 900 RE have been isolated from over 230 species of bacteria

30. Why don’t bacteria destroy their own DNA with their restriction enzymes?

31. Methylation

32. Special characteristics of RE:1. The restriction site sequences recognized by most restriction enzymes are pallindromes.2. Most restriction enzymes are highly specialized to such an extent that they recognize only a single restriction site. 3. The restriction site is recognized irrespective of the DNA source.

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34. Some concepts:Different restriction enzymes that recognize the same sequence are known as neoschizomers. These often cleave in a different locales of the sequence; however, different enzymes that recognize and cleave in the same location are known as an isoschizomer.Isocaudomers are enzymes that recognize slightly different sequences but produce the same ends.

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