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RDT
- 1. JIWAJI UNIVERSITY CENTER FOR GENOMICS MOLECULAR & HUMAN GENETICS Presented by:- AKANSHA CHAUHAN 3rd SEMESTER
- 3. CONTENTS Introduction Principle of rDNA technology Applications of rDNA technology Restriction Endonucleases enzyme DNA Ligase DNA Polymerase DNA Modifying enzymes
- 4. Introduction Recombinant DNA Technology is the joining together of DNA molecules from two different species. The Recombinant DNA molecule is inserted into a host organism to produce new genetic combinations. Recombinant DNA technology also known as Gene Cloning/Molecular Cloning, Genetic Engineering, Genetic modification. The first recombinant molecule containing DNA from different organisms was assembled late in 1971. The technology for propagating and expressing recombinant genes was invented by Stanley Cohen and Herbert Boyer in 1973.
- 5. Basic principles of rDNA technology Generation of DNA fragments & selection of the desired piece of DNA. Insertion of the selected DNA into a cloning vector to create rDNA. Introduction of the recombinant vectors into host cells. Multiplication & selection of clones containing the recombinant molecules. Expression of the gene to produce the desired product.
- 7. Applications of Recombinant DNA Technology rDNA technology has a wide range of application in industries, medical science, & agriculture as “well as molecular biology”. Molecular diagnosis of diseases Gene therapy DNA fingerprinting Production of vaccines Commercial & pharmaceutical products.
- 8. Enzymes used for rDNA Technology rDNA technology produce rDNA using a set of enzymes known as recombinant enzymes. They are :- 1. Nucleases 2. Ligases 3. Polymerases 4. DNA modifying enzymes
- 9. Restriction Endonucleases Enzymes Restriction endonucleases are indispensable for DNA cloning & sequencing. They serve as the tools for cutting DNA molecules at predetermined sites, which is the basic requirement for gene cloning or rDNA technology. The presence of restriction enzymes was postulated by W. Arber, during 1960s, while the first true restriction endonuclease was isolated in 1970 by Smith, Nathans. Nucleases are enzymes that degrade DNA molecules by breaking the phosphodiester bonds. Nucleases are two types:- Exonuclease : removes the terminal nucleotide of the DNA molecule
- 10. Continued Endonucleases : breaks the internal phosphodiester bond. Endonucleases are the most widely used ones. Restriction Endonucleases were first discovered in E.coli. There are four distinct types of restriction endonucleases: i. Type I ii. Type II iii. Type III iv. Type IV
- 11. Feature Type I Type II Type III Type IV Structural subunits Three different Two identical Two different Two different Enzyme activity Endonuclease, Methyltransferas e ATPase Endonuclease or methyltransferase Endonuclease, methyltransferase ATPase Endonuclea se GTPase Biochemical cofactors for DNA cleavage ATP, AdoMet, Mg2+ Mg2+ ATP, Mg2+ Mg2+, GTP Methylation AdoMet, Mg2+ AdoMet AdoMet, Mg2+ — Recognition sequence Asymmetric, bipartile Usually symmetric Asymmetric Bipartile, methylated Cleavage site Random, atleast 1000bp from recognition site At or near recognition site 25-27bp from recognition site Between methylated bases at multiple positions DNA translocation Yes No Yes Yes
- 12. Nomenclature The nomenclature of restriction endonucleases follows a general pattern. The 1st letter of the name of genus in which a given enzyme is discovered is written in capital. This is followed by the first two letters of species name of the organism. These three letters are written in italics, as Eco from Escherichia coli, Hin from Haemophilus influenzae. EcoRI :- E = genus, co = species, R = strain(Ry13), I= order of discovery Ist .
- 13. Recognition Sequence It is a site where DNA is cut by Restriction endonuclease. It is 4-8bp in sequence. It is pallindromic in sequence. Example- ROTATOR 5’GAATTC3’ 3’CTTAAG5’ Most Type II restriction endonucleases have recognition sites of 4, 5, or 6bp, which are GC – rich. EcoRI recognition site 5’GAA TTC3’ 3’CTT AAG5’
- 14. Mode of action of Restriction Enzyme Restriction enzymes cuts DNA molecules in two different styles. Sticky or Staggered style • Most common cutting by Restriction Endonucleases • Complementary ends present. • Examples- EcoRI, BamHI EcoRI - 5’GAATTC3’ 5’GAATTC3’ 3’CTTAAG5’ 3’CTTAAG5’ BamHI - 5’GGATCC3’ 5’GGATCC3’ 3’CCTAGG5’ 3’CCTAGG5’
- 15. Continued Blunt Style • Less common used in RDT • No complementary ends present. • Example HaeIII, AluI HaeIII- 5’GG CC3’ 3’CC GG5’ AluI - 5’AG CT3’ 3’TC GA5’
- 16. DNA Ligase DNA ligase act as a molecular glues as they help in sealing gaps in DNA fragments. In 1969 Har Govind Khorana discovered DNA ligase in T4 bacteriophage. The enzyme used most often in RDT is T4DNA ligase. DNA ligase joins two ends of DNA & help in the synthesis of phosphodiester bond. This phosphodiester bond is formed between 3’OH group at the end of 1 polynucleotide chain & 5’phosphate group at the end of other polynucleotide chain of DNA. This enzyme changes linear DNA into circular DNA & helps in formation of recombinant molecule.
- 17. Continued Restriction digestion of DNA generates two types of DNA ends- sticky ends or blunt ends. Blunt ends are simple direct and non-cohesive. Special types of DNA ligase are used to ligate these types of DNA ends. Sticky ends are cohesive & have few basepairs palindromic sequences on both the strands. Sticky ends are generated for inserting a gene of interest in the plasmid. It works better in comparison with the blunt end ligation.
- 18. DNA Ligase Sticky end Blunt end researchgate.co geneticeducation.co.in
- 19. DNA Polymerase DNA polymerase is a complex enzyme which synthesize nucleotide complementary to template strand. It adds nucleotide to free 3’OH end and help in elongation of strand. It also helps to fill gap in double stranded DNA. DNA polymerase was first discovered by Kornburg in 1959 in E.coli bacteria. Taq polymerase isolated from Thermus aquaticus is used in PCR. There are three types of DNA polymerases:-PolymeraseI, PolymeraseII, DNA polymeraseIII.
- 20. continued The DNA polymeraseI enzyme, apart from addition of single nucleotide to free OH end of DNA chain, also catalyzes 3’ 5’ & 5’ 3’ exonuclease activity. DNA polymerasesII also catalyzes 3’ 5’ exonucleases activity. The polymerization activity of polymeraseII is less than the polymeraseI & about 50 nucleotides per minute are synthesized. DNA polymerasesIII requires an auxiliary protein DNA copolymerase III for its polymerization activity. It can polymerize 15000 nucleotide per minute.
- 22. DNA modifying enzymes There are numerous enzymes that modify DNA molecules by addition or removal of specific chemical groups. The most important are as:- Alkaline phosphatase Polynucleotide kinase Terminal deoxynucleotidyl transferases
- 23. Alkaline phosphatase • It is isolated from E.coli or calf intestinal tissue. • It removes the phosphate group present at 5’terminal of the DNA molecule. • This enzyme is used to stop the ligation reaction between the DNA molecules. biologyexams4u.com
- 24. Polynucleotide kinase It isolated from E.coli which has reverse effect of alkaline phosphates add ℗ terminal in the end. It uses ATP as phosphate donor in reaction It promotes ligation reaction brainkart.com
- 25. Terminal deoxynucleotidyl transferases This enzyme is responsible for Homopolymer tailing. This enzyme involves the addition of oligo to 5’ends of DNA & addition of oligo to 3’end of DNA. The complementary end of DNA can be joined by annealing process. The tailing is catalyzed by enzyme (ToT) biologyexams4u.com
- 26. Conclusion Recombinant DNA technology is an important development in science that has made the human life much easier. It has advanced strategies for cancer treatment, genetic diseases, diabetes. This technology also involved in forensic science. The combination of restriction modification enzymes representing cutting and joining functions in DNA manipulation and genetic engeeniring enable the application of a wide variety of procedures.
- 27. References nptel.ac.in onlinebiologynotes.com sciencedirect.com prezi.com britannica.com biologynotes4u.com brainkart.com