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Introduction of Biotechnology. Chapter 7 Lecture 7-3 Microbial Genetic Engineering. Topics Useful Tools and Techniques Recombinant DNA Technology Recombinant microorganisms Genome Analysis Practical applications. Tools and Techniques. Enzymes as Tools DNA analysis

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Chapter 7 Lecture 7-3 Microbial Genetic Engineering

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    1. Introduction of Biotechnology Chapter 7Lecture 7-3 Microbial Genetic Engineering Topics Useful Tools and Techniques Recombinant DNA Technology Recombinant microorganisms Genome Analysis Practical applications

    2. Tools and Techniques • Enzymes as Tools • DNA analysis • Nucleic acid hybridization • Synthesizing DNA • Polymerase Chain Reaction

    3. Enzymes • Restriction endonuclease • Ligase • Reverse transcriptase • cDNA

    4. Restriction endonuclease • Originates in bacterial cells • Many different types exist • Natural function is to protect the bacterium from foreign DNA (bacteriophage) • Recognizes 4 to 10 base pairs (palindromic sequence) • Cleaves DNA at the phosphate-sugar bond • Used in the cloning method

    5. The function of a restriction endonuclease or enzyme. Fig. 2.3.1 Some useful properties of DNA

    6. Ligase (EC6.5.1.1) • Link DNA fragments • Rejoin the phosphate-sugar bonds • Used often genes into vectors in the cloning method

    7. Reverse transcriptase • Converts RNA to DNA • Ex. Complementary DNA (cDNA) • Required for eucaryote gene expression • mRNA to cDNA • No introns are present

    8. Fig. 2.3.2 Synthesis of double-stranded cDNA from mRNA

    9. Analysis of DNA • Electrophoresis • Hybridization and probes • Sequencing • Polymerase Chain Reaction

    10. Electrophoresis • Separation of DNA on size • Negative charge DNA (phosphate group) migrates to positive electrode • Usefulness • Characterizing DNA fragment • Fingerprinting

    11. Steps with the electrophoresis technique. Fig. 2.3.3 Revealing the patterns of DNA with electrophoresis

    12. Hybridization and probes • Complementation • Probes • Southern Blot

    13. Complementation • Complementary sites on two different nucleic acids bind or hybridize • DNA hybridize with DNA • DNA hybridize with RNA • RNA hybridize with RNA

    14. Probes • Small stretches of nucleic acid with a known sequence called an oligonucleotide • Single stranded • Detects specific nucleotide sequences in unknown nucleic acid samples

    15. Alternate hybridization methods can be used to detect unknown bacteria or virus. Fig. 2.3.4 A hybridization test using microbe-specific probes

    16. Southern blot • Method for detecting an unknown sample of DNA • Incorporates restriction endonuclease, electrophoresis, denaturing, probing, and visual detection.

    17. A Southern blot separates DNA by electrophoresis, denatures and transfers the DNA to filter paper, and uses probes to visualize hybridization. Fig. 2.3.5 Conducting a Southern blot hybridization test.

    18. Sequencing • Provide the identity and order of nucleotides (bases) • Method • Sanger method Fig. 2.3.6 Dideoxyadensine triphosphate (ddATP)

    19. The Sanger method of sequencing DNA. Fig. 2.3.7 Steps in a Sanger DNA sequence technique

    20. Polymerase Chain Reaction (PCR) • Specific amplification of DNA • Involves a denaturing, priming (annealing), and extension cycle • 30 cycles are sufficient for detection of DNA • Can be used to detect disease or infectious agents

    21. A schematic of the PCR reaction and its products Fig. 2.3.8 Diagram of the polymerase chain reaction

    22. Recombinant DNA • Recombinant • Cloning vectors • Cloning host • Applications

    23. Recombinant • When a cloning host receives a vector containing the gene of interest • A single cloning host containing the gene of interest is called a clone

    24. Practical applications of recombinant technology include the development of pharmaceuticals, genetically modified organisms, and forensic techniques. Fig. 2.3.9 Methods and applications of genetic technology

    25. Cloning vectors • Carry a significant piece of the donor DNA (gene of interest) • Readily accepted DNA by the cloning host • Contain an origin of replication • Contain a selective antibiotic resistant gene • Ex. Plasmids, phages, microbial artificial chromosomes

    26. An example of a plasmid vector. Fig. 2.3.10 Partial map of the pBR322 plasmid of E. coli

    27. An example of a cosmid vector. Fig. 2.3.11 Plasmid map of the pJB8 cosmid of E. coli

    28. An example of a yeast artificial chromosome. Fig. 2.3.12 Yeast artificial chromosome with inserted human DNA

    29. Cloning host • Bacteria (prokaryote) • Ex. Escherichia coli • Bacteria will not excess introns from eukaryotic DNA • Fungi (eukaryote) • Ex. Saccharomyces cerevisiae • Will excess introns

    30. Applications • Protein production • Alter organisms normal function • Source of DNA (synthesis)

    31. Biochemical Products of Recombinant DNA Technology Enables large scale manufacturing of life-saving hormones, enzymes, vaccines insulin for diabetes human growth hormone for dwarfism erythropoietin for anemia Factor VIII for hemophilia HBV vaccine 31

    32. Fig. 2.3.13 Cloning and production of recombinant somatostatin in E. coli

    33. Important protein products generated by recombinant DNA technology. Table 2.1 Current protein products from recombinant DNA technology

    34. Recombinant Organisms • Modified bacteria and viruses • Transgenic plants • (Transgenic animals)

    35. Modified bacteria • Pseudomonas syringae • Prevents frost crystals from forming on plants • Pseudomonas fluorescens • Contains an insecticide gene

    36. The construction of a recombinant in order to produce the human alpha-2a interferon. Fig. 2.3.14 Steps in recombinant DNA, gene cloning, and product retrieval.

    37. Transgenic plants • Agrobacterium tumefaciens • Ti plasmid contains gene of interest, and is integrated into plant chromosome • Ex. tobacco, garden pea, rice

    38. Schematic of A. tumefaciens transferring and integrating the Ti plasmid into the plant chromosome. Fig. 2.3.15 Bioengineering of plants

    39. Antisense and triplex DNA • Antisense RNA or DNA • Prevent the synthesis of an unwanted protein • Targets mRNA • Triplex DNA • Prevents transcription • Targets double stranded DNA

    40. Examples of the mechanism for antisense DNA and triplex DNA. Fig. 2.3.16 Mechanisms of antisense DNA and triplex DNA

    41. Genome Analysis • Maps • Fingerprints • Family trees

    42. Maps • Determine the location of particular genes (locus) on the chromosome • Determine differences in chromosomal regions (alleles) • Types of maps • Genomics and bioinformatics

    43. Types of maps • Linkage • Shows the relative proximity and location of genes • Physical • Shows the proximity and size of genes • Sequence • Shows the exact order of bases

    44. Fig.2.3.17 Map of Haemophilus influenzae genome

    45. Genomic and bioinformatics • New discipline of study as a result of the enormous data generated by maps • Analyze and classify genes • Determine protein sequences • Determine the function of the genes

    46. Fingerprinting • Emphasizes the differences in the entire genome • Techniques • Endonucleases • PCR • Southern blot • Uses • Forensic medicine • Identify hereditary disease

    47. Comparing the fingerprints for different individuals. Fig. 2.3.18 DNA fingerprints:the bar codes of life

    48. Family trees • Determine the genetic family tree for many diseases • Pedigree of domestic animals • Genetic diversity in animals

    49. Genetic screening for Alzheimer’s disease. Fig. 2.3.19 Pedigree analysis based on genetic screening

    50. The microarray technique is a way of detecting gene expression from different individuals. Fig. 2.3.20 Gene expression analysis using microarrays