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Genetic Engineering andRecombinant DNA TechnologyPresented byDr. B.Victor., Ph.D.Email: bonfiliusvictor @gmail.comBlog: bo...
 Genetic engineering-                   definition, objectives and basicPresentation       methodologyout line          ...
Genetic engineeringDefinition -1• A set of techniques capable to allow the  identification, manipulation and multiplicatio...
Objectives of Genetic Engineering Basic research on gene structure and function Production of useful products and services...
GeneticengineeringBasic methodology
Recombinant DNA technology    r DNA technology    • The technology of preparing      r DNA in vitro by cutting up      DNA...
Meaning r DNA technology    Modifying the genetic    make up an organism    by:    • Adding new genes    • Changing the ex...
Objectives of    Artificially synthesize newr DNA               genes.Technology                   Altering the genome o...
Discovery of DNA structure-              Watson and Crick in 1953                 Isolation of DNA ligase in 1967History o...
Biological tools of r DNA technology          Foreign DNA/    Vehicle/vector                 Culture media,Enzymes        ...
 DNA or RNA polymerase-Chemical              replicating or annealing a DNAknives in             chain.molecular         ...
 A special class of sequence –Restriction                    specific enzymesendonucleases                   Found in ba...
   Recognition sequences of RE ases                    are palindromesRestriction                       A palindrome is ...
Salient features                                         Bacterialof Restriction     Endonucleases                        ...
 A map showing the unique sitesRestriction     of cutting of the DNA of amapping         particular organism by a single ...
 Preparing a restriction mapApplications of                        of DNARestrictionendonucleases          Fragmenting g...
Vector DNA or                               Plasmid DNAVehicle DNA                       Bacterial                        ...
 Types of VectorsTypes of      Bacterial plasmid vectorsvectors       Bacteriophage vectors              Cosmid vector...
 Capable of autonomous                    replication independent of theCharacteristics     main bacterial chromosomeof a...
   Size                 Origin of replication (ori)                 Multiple cloning site (MCS)Practical        Select...
 Plasmid DNA – small               circular DNA found inPlasmids       bacteria              They replicate             ...
 Two types of phage vectorsPhage Vectors     have been extensively                  developed-λ and M13.                ...
23           Hybrid vector constructed to            contain features from bothCosmids     phages and plasmids.          ...
24               large fragments of DNA canArtificial        be cloned.Chromosomes                 Mapping of genes is e...
Host cell types            Host cell types  Prokaryotic                  Eukaryotic     hosts                       hosts ...
Two types of host-vectors Cloning            • Propagation of  vector      DNA insertsExpression           • Production of...
Isolate desired                   DNAMaking of            Cut with a suitabler DNA             REase            Ligate int...
Step 1– fragmentation -                    breaking apart a strand of                    DNAMolecularCloning /DNA cloning ...
Recombinant DNA cloning procedure                                        Screening of            FineForeign              ...
1. Choice of host organisms                  and cloning vectorDNA             2.   Preparation of vector DNAcloning      ...
Gene transfer technologyTransduction      • Virus mediated gene transfer Tranfection      • Chemical or physical tricks to...
   This method was described by                        Graham and Van der Eb in 1973.Calcium                A process fo...
   It involves a brief application of                      high voltage electric current toElectroporation       the cell...
Selectiontechniques for       DNArDNA                               Colony                 hybridizationmolecules         ...
 After transformation, theProcess of               bacteria are challenged withselection                an antibiotic (su...
 This technique was                  introduced by Grunstein andDNAhybridization                  Hogness (1978).assay   ...
 The transformed colonies are                  transferred to a nitrocellulose filter.Colony           The colonies are ...
The Tools of                  Gene Libraries   Recombinant                     Collections of cloned DNA   DNA Technolog...
   contains only complementary DNA                molecules synthesized from mRNA                molecules in a cell.cDNA...
 Multiplying DNA in vitro: The   Techniques of                 Polymerase Chain Reaction   Recombinant                   ...
 Separating DNA Molecules: Gel   Techniques of   Recombinant                                 Electrophoresis   DNA Techno...
Techniques of                 Separating DNA Molecules:   Recombinant                   Gel Electrophoresis and the   DNA...
Techniques of                 DNA Microarrays   Recombinant                     Consist of molecules of   DNA Technology...
   In situ hybridization                     Used to determine the cell type thatApplications          is expressing the...
 Studying Gene Expression                      Gene microarrays                        DNA microarray analysis         ...
Applications of               Genetic Mapping   Recombinant                   Provides knowledge of total   DNA Technolo...
Applications of               Pharmaceutical and   Recombinant                   Therapeutic Applications   DNA Technolog...
 Agricultural Applications   Applications of   Recombinant                      Production of transgenic organisms   DNA...
Applications of                                 Agricultural Applications   Recombinant                     Pest resista...
Applications of                                 Agricultural Applications   Recombinant                     Improvements...
   Started in 1990 by the U.S.                Department of EnergyThe Human      International collaborative effortGenom...
Medical products made from rDNA tech  Product                year    treatmentHuman insulin            1982   diabetessoma...
   Dr.B.Victor is a highly                experienced professor, recently                retired from the reputed        ...
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Genetic engineering and recombinant DNA technology

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Genetic Engineering is a set of techniques capable to allow the identification, manipulation and multiplication of genes of living organisms.

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Genetic engineering and recombinant DNA technology

  1. 1. Genetic Engineering andRecombinant DNA TechnologyPresented byDr. B.Victor., Ph.D.Email: bonfiliusvictor @gmail.comBlog: bonvictor.blogspot.com
  2. 2.  Genetic engineering- definition, objectives and basicPresentation methodologyout line  Recombinant DNA technology- definition, objectives, history and enzyme tools.  Restriction enzymes - features, types of cuts.  Restriction mapping- applications  Vector DNA – types, characteristics and practical features  Host cell types  Recombinant DNA technology- procedure  Gene transfer technology  Screening technology  Applications
  3. 3. Genetic engineeringDefinition -1• A set of techniques capable to allow the identification, manipulation and multiplication of genes of living organisms.Definition -2• Changing of genes by using in vitro processes.Other names• Gene manipulation, gene cloning• Recombinant DNA technology, genetic modification
  4. 4. Objectives of Genetic Engineering Basic research on gene structure and function Production of useful products and services Generation of transgenic plants and animals Investigation of human genome for gene therapy
  5. 5. GeneticengineeringBasic methodology
  6. 6. Recombinant DNA technology r DNA technology • The technology of preparing r DNA in vitro by cutting up DNA molecules and splicing them together fragments from more than one organism.
  7. 7. Meaning r DNA technology Modifying the genetic make up an organism by: • Adding new genes • Changing the existing genes
  8. 8. Objectives of  Artificially synthesize newr DNA genes.Technology  Altering the genome of an organism.  Bring about new gene combinations not found in nature.  Understanding the hereditary diseases and their cure.  Improving human genome.
  9. 9. Discovery of DNA structure- Watson and Crick in 1953 Isolation of DNA ligase in 1967History ofrecombinantDNA Isolation of REase in 1970technology Jackson, Symons, and Berg (1972) generated first recombinant DNA molecules. Cohen and Boyer (1973) produced first plasmid vector capable of being replicated within a bacterial host
  10. 10. Biological tools of r DNA technology Foreign DNA/ Vehicle/vector Culture media,Enzymes Host cells Synthetic DNA DNA Buffers, reagents
  11. 11.  DNA or RNA polymerase-Chemical replicating or annealing a DNAknives in chain.molecular  Reverse transcriptase – synthesizecarpentry c DNA from RNA template.Enzymes are  DNA ligase – joining DNA strandschemical knives together.in r DNA  Nuclease-breaks phospho-diestertechnology bonds within free ends (exonucleases) or in an interior position( Endonucleases ).  Restriction endonuclease – recognize a specific base sequence and cuts the DNA.
  12. 12.  A special class of sequence –Restriction specific enzymesendonucleases  Found in bacteria which protect its(RE ases) genetic material from the invasive attacks of viruses.  Site-specific - cleave DNA molecules only at specific nucleotide sequences.  REases recognize DNA base sequences that are palindromes.  REases make two single stranded breaks, one in each strand.  RE ases make staggered cuts with complementary base sequences for easy circularization.
  13. 13.  Recognition sequences of RE ases are palindromesRestriction  A palindrome is a word, phrase,Enzymes- number or other sequence of unitsTypes of cuts that can be read the same way in either direction  Cohesive (sticky) ends – overhanging single-stranded ends  Blunt ends – double-stranded, non-overhanging ends
  14. 14. Salient features Bacterialof Restriction Endonucleases enzymesendonucleases(RE ases) Different REases Names of REases Isolated from are derived different from source bacteria bacteria. Cut DNA into Basic tool of defined and Genetic reproducible fragments Engineer
  15. 15.  A map showing the unique sitesRestriction of cutting of the DNA of amapping particular organism by a single REase enzyme.  A particular REase generates a unique set of DNA fragments with specific base sequence.  Another enzyme will generate a different set of DNA fragments from the same DNA molecule.  The family of DNA fragments generated by a single enzyme can be detected easily gel electrophoresis.
  16. 16.  Preparing a restriction mapApplications of of DNARestrictionendonucleases  Fragmenting genomic DNA(RE ases) prior to Southern Blotting.  Generating DNA fragments RE ases catalyze that can be sub-cloned in sequence – appropriate vectors. dependent double- stranded breaks in  Generating DNA fragments DNA yielding a for labeled probes homogeneous population of DNA fragments.
  17. 17. Vector DNA or Plasmid DNAVehicle DNA Bacterial Bacteriophage DNAs DNAThe DNA which Vectoracts as a carrier is DNAa vehicle DNA Viral DNAs Yeast DNAs
  18. 18.  Types of VectorsTypes of  Bacterial plasmid vectorsvectors  Bacteriophage vectors  Cosmid vectors  Expression vectors  Bacterial Artificial Chromosomes (BAC)  Yeast Artificial Chromosomes (YAC)  Ti and Ri vectors
  19. 19.  Capable of autonomous replication independent of theCharacteristics main bacterial chromosomeof anprokaryotic  Easy to isolate, i.e. small.vector  Non -toxic to host cells.  Have space for foreign inserts.  Have unique restriction sites for common restriction enzymes.  Have convenient markers for selection of transformants, e.g. antibiotic resistance genes  Be relaxed, i.e. multiple copies in a host cell.
  20. 20.  Size  Origin of replication (ori)  Multiple cloning site (MCS)Practical  Selectable marker genesFeatures of  RNA polymerase promoterDNA Cloning sequencesVectors  DNA sequencing primers
  21. 21.  Plasmid DNA – small circular DNA found inPlasmids bacteria  They replicate autonomously.  Easily purified  Confer antibiotic resistance to host bacteria –allow easy identification.  First type of cloning vector developed.
  22. 22.  Two types of phage vectorsPhage Vectors have been extensively developed-λ and M13.  phage vectors have engineered phage genomes previously genetically modified to include restriction sites.  after insertion of foreign DNA, the recombinant phage genome is packaged into the capsid and used to infect host cells
  23. 23. 23  Hybrid vector constructed to contain features from bothCosmids phages and plasmids.  Cosmids have a selectable marker, multiple cloning sites from plasmids and a cos site from l phage
  24. 24. 24  large fragments of DNA canArtificial be cloned.Chromosomes  Mapping of genes is easier.  One copy of YAC is present per cell.  yeast artificial chromosomes (YACs)  bacterial artificial chromosomes (BACs)  played important role in the human genome project
  25. 25. Host cell types Host cell types Prokaryotic Eukaryotic hosts hosts Yeast Bacteria Algae E . Coli fungi Bacillus sp. Yeast - SaccharomycesPseudomonas sp. Fungi- Aspergillus, NeurosporaStreptomyces sp. Algae - Chlamydomonas
  26. 26. Two types of host-vectors Cloning • Propagation of vector DNA insertsExpression • Production of vector proteins
  27. 27. Isolate desired DNAMaking of Cut with a suitabler DNA REase Ligate into a suitable cloning vector Transform r DNA into a suitable host cell
  28. 28. Step 1– fragmentation - breaking apart a strand of DNAMolecularCloning /DNA cloning Step 2 – ligation-gluing together pieces of DNA in a desired sequence.Molecular cloningrefers to the Step 3 –Transfection -process of making inserting the newly formed DNA into cells.multiple DNAmolecules. Step 4-Screening / selection – selecting out the cells that were successfully tranfected with the new DNA
  29. 29. Recombinant DNA cloning procedure Screening of FineForeign Transfection transformants chemicals DNA Hormones Host Desired r DNA Enzymes cell products vaccinesPlasmid Antibiotics DNA Antibodies Blood factors
  30. 30. 1. Choice of host organisms and cloning vectorDNA 2. Preparation of vector DNAcloning 3. Preparation of DNA to beprotocol – cloned7 steps 4. Creation rDNA. 5. Introduction of rDNA into the host organism. 6. Selection of organisms containing rDNA. 7. Screening for clones with desired DNA inserts and biological properties.
  31. 31. Gene transfer technologyTransduction • Virus mediated gene transfer Tranfection • Chemical or physical tricks to persuade cells to take DNA from the culture medium • Physically inserting the geneDirect transfer • e.g. microinjectionNatural gene • A receptor – mediated lateral binding • Fusogenic proteins used transfer
  32. 32.  This method was described by Graham and Van der Eb in 1973.Calcium  A process for inserting foreignphosphate – DNA into bacteriaco precipitate  Treat bacterial cells with ice-coldmethod calcium chloride  Add plasmid DNA to cells chilled on ice which form calciumTransformation of phosphate –DNA precipitate.Bacterial Cells  Heat the cell and DNA mixture to 42oC  Membrane becomes fluid and plasmid DNA enters bacterial cells and is replicated and expressed
  33. 33.  It involves a brief application of high voltage electric current toElectroporation the cells resulting in the formation of transient holes in the cell membrane through which plasmid DNA can enter the cell.  The transformation efficiency is high.  Quick restoration of membrane fluidity and closing of pores is crucial for survival of cell after the pulse.
  34. 34. Selectiontechniques for DNArDNA Colony hybridizationmolecules immunoassay assay Screening by Genetic protein screening activity methods
  35. 35.  After transformation, theProcess of bacteria are challenged withselection an antibiotic (such as ampicillin).  If the E. coli have taken up and expressed an ampicillin resistance gene on aSelection is a plasmid, they will live -process designed to otherwise they will die.facilitate theidentification of  This process is calledrecombinant bacteria selection because selectedwhile preventing thegrowth of non- bacteria may survive.transformedbacteria.
  36. 36.  This technique was introduced by Grunstein andDNAhybridization Hogness (1978).assay  The target DNA is denatured at 800C and bound to a nitrocellulose filter discs.  Such filters are hybridized with radioactive DNA probes.  The results are monitored by autoradiography.
  37. 37.  The transformed colonies are transferred to a nitrocellulose filter.Colony  The colonies are lysed and the releasedimmunoassay proteins are attached to the matrix.  The matrix is treated with a primary antibody which specifically binds to the proteins encoded by the target gene.  Then the matrix was washed to remove any unbound antibody. Then the matrix was treated with a second antibody which was an enzyme, alkaline phosphatase.  the target protein (antigen)was treated with a colorless substrate.  The colorless substrate is hydrolysed by the alkaline phosphatase into a colored complex.
  38. 38. The Tools of  Gene Libraries Recombinant  Collections of cloned DNA DNA Technology fragments from a particular organism contained within bacteria or viruses as the host  Library may contain all genes of a single chromosome  Screening, identification and characterization of cloned fragments are possible with suitable probes.© 2012 Pearson Education Inc.
  39. 39.  contains only complementary DNA molecules synthesized from mRNA molecules in a cell.cDNA  mRNA from tissue of interest isLibraries isolated  Converted to a double-stranded DNA by using the enzyme reverse transcriptase  Called complementary DNA (cDNA) because it is an exact copy of the mRNA
  40. 40.  Multiplying DNA in vitro: The Techniques of Polymerase Chain Reaction Recombinant (PCR) DNA  Developed in the 1980s by Kary Mullis Technology  Technique for making copies, or amplifying, a specific sequence of DNA in a short period of time  Repetitive process consisting of three steps  Denaturation  Priming  Extension  Can be automated using a thermocycler  At the end of one cycle, the amount of DNA has doubled  Cycles are repeated 20–30 times© 2012 Pearson Education Inc.
  41. 41.  Separating DNA Molecules: Gel Techniques of Recombinant Electrophoresis DNA Technology  Gel electrophoresis  Separates molecules based on electrical charge, size, and shape  Allows to isolate DNA of interest  Negatively charged DNA drawn toward positive electrode  Agarose makes up gel; acts as molecular sieve  Smaller fragments migrate faster than larger ones  size is determined by comparing distance migrated to standards© 2012 Pearson Education Inc.
  42. 42. Techniques of  Separating DNA Molecules: Recombinant Gel Electrophoresis and the DNA Technology Southern Blot  Southern blot  DNA transferred from gel to nitrocellulose membrane  Probes used to localize DNA sequence of interest  Uses of Southern blots  Genetic “fingerprinting”  Diagnosis of infectious disease© 2012 Pearson Education Inc.
  43. 43. Techniques of  DNA Microarrays Recombinant  Consist of molecules of DNA Technology immobilized single-stranded DNA  Fluorescently labeled DNA washed over array will adhere only at locations where there are complementary DNA sequences  Uses of DNA microarrays  Monitoring of gene expression  Diagnosis of infection  Identification of organisms in an environmental sample© 2012 Pearson Education Inc.
  44. 44.  In situ hybridization  Used to determine the cell type thatApplications is expressing the mRNAof  Tissue of interest is preserved in aRecombinant fixative solution and embedded in a wax-like substanceDNA  Tissue can be sliced into very thinTechnology - sections attached to microscopeStudying Gene slidesExpression  Slides are incubated with a probe to the gene of interest  Probe hybridizes with mRNA in cells  Probe is detected
  45. 45.  Studying Gene Expression  Gene microarrays  DNA microarray analysis  Single-stranded DNA moleculesApplications of are attached onto a slide using aRecombinant robotic arrayer fitted with tiny pinsDNA  Can have over 10,000 spots of DNATechnology  Extract mRNA from tissue of interest, tag it with fluorescent dye, and incubate overnight with the slide  mRNA will hybridize to spots on the microarray that have complimentary DNA sequences  Slide is scanned with a laser that causes the spots to fluoresce
  46. 46. Applications of  Genetic Mapping Recombinant  Provides knowledge of total DNA Technology number of all genes.  Shows relationships between genes.  Provides all genetic information about the organism.© 2012 Pearson Education Inc.
  47. 47. Applications of  Pharmaceutical and Recombinant Therapeutic Applications DNA Technology  Gene therapy  Missing or defective genes replaced with normal copies  Medical diagnosis  Patient specimens can be examined for presence of gene sequences unique to certain pathogens  Xenotransplants  Animal cells, tissues, or organs introduced into human body© 2012 Pearson Education Inc.
  48. 48.  Agricultural Applications Applications of Recombinant  Production of transgenic organisms DNA Technology  Recombinant plants and animals altered by addition of genes from other organisms  Herbicide tolerance  Gene from Salmonella conveys resistance to glyphosate (Roundup™) Farmers can kill weeds without killing crops  Salt tolerance  Scientists have inserted gene for salt tolerance into tomato and canola plants  Transgenic plants survive, produce fruit, and remove salt from soil© 2012 Pearson Education Inc.
  49. 49. Applications of  Agricultural Applications Recombinant  Pest resistance DNA Technology  Bt toxin  Naturallyoccurring toxin harmful only to insects  Gene for Bt toxin inserted into various crop plants  Genes for Phytophthora resistance inserted into potato crops© 2012 Pearson Education Inc.
  50. 50. Applications of  Agricultural Applications Recombinant  Improvements in nutritional value DNA Technology and yield  Tomatoes allowed to ripen on vine and shelf life increased  Genefor enzyme that breaks down pectin suppressed  BGH allows cattle to gain weight more rapidly  Produce meat with lower fat content and produce 10% more milk  Gene for β-carotene inserted into rice© 2012 Pearson Education Inc.
  51. 51.  Started in 1990 by the U.S. Department of EnergyThe Human  International collaborative effortGenome to identify all human genes and to sequence all the base pairs of theProject 24 human chromosomes  20 centers in 6 countries: China, France, Germany, Great Britain, Japan, and the United States.  April 14, 2003, map of the human genome was completed  Consists of 20,000 to 25,000 protein-coding genes
  52. 52. Medical products made from rDNA tech Product year treatmentHuman insulin 1982 diabetessomatotropin 1985 Pituitary dwarfismHepatitis B vaccine 1986 Immunization for hepatitis B virusErythropoietin 1988 anemiaInterleukin-2 1989 Cancer of kidneyWhooping cough vaccine 1989 Immunization for Whooping coughFactor VIII 1993 hemophilia
  53. 53.  Dr.B.Victor is a highly experienced professor, recently retired from the reputed educational institution- St. Xavier’ sAbout the College, Palayamkottai, India-Presenter 627001.  He was the dean of sciences, IQAC coordinator and assistant controller of examinations.  He has more than 32 years of teaching and research experience  He has taught a diversity of college courses and guided 12 PhDs.  Send your comments to : bonfiliusvictor@gmail.com
  54. 54. Thank a lot for your watching

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