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Foundations in Microbiology. PowerPoint to accompany. Fifth Edition. Talaro. Chapter 10. Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Genetic Engineering: A Revolution in Molecular Biology. Chapter 10. Genetic engineering.

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foundations in microbiology

Foundations in Microbiology

PowerPoint to accompany

Fifth Edition




Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

genetic engineering
Genetic engineering
  • direct, deliberate modification of an organism’s genome
  • bioengineering
  • Biotechnology – use of an organism’s biochemical and metabolic pathways for industrial production
i tools techniques of genetic engineering
I. Tools & Techniques of genetic engineering
  • enzymes for dicing, splicing, & reversing nucleic acids
  • analysis of DNA
enzymes for dicing splicing reversing nucleic acids
Enzymes for dicing, splicing, & reversing nucleic acids
  • restriction endonucleases – recognize specific sequences of DNA & break phosphodiester bonds
  • ligase – rejoins phosphate-sugar bonds cut by endonucleases
  • reverse transcriptase – makes a DNA copy of RNA - cDNA
analysis of dna
Analysis of DNA
  • gel electrophoresis- separates DNA fragments based on size
  • nucleic acid hybridization & probes – probes base pair with complementary sequences; used to detect specific sequences
  • DNA Sequencing – reading the sequence of nucleotides in a stretch of DNA
  • Polymerase Chain Reaction – way to amplify DNA
ii methods in recombinant dna technology
II. Methods in Recombinant DNA Technology
  • concerned with transferring DNA from one organism to another
  • Cloning vectors & hosts
  • Construction of a recombinant plasmid
characteristics of cloning vectors
Characteristics of cloning vectors
  • must be capable of carrying a significant piece of donor DNA
  • must be readily accepted by the cloning host
  • plasmids – small, well characterized, easy to manipulate & can be transferred into appropriate host cells through transformation
  • bacteriophages – have the natural ability to inject their DNA into bacterial hosts through transduction
vector considerations
Vector considerations
  • origin of replication
  • size of donated DNA vector will accept
  • gene which confers drug resistance to their cloning host
characteristics of cloning hosts
Characteristics of cloning hosts
  • rapid overturn, fast growth rate
  • can be grown in large quantities using ordinary culture methods
  • nonpathogenic
  • genome that is well delineated
  • capable of accepting plasmid or bacteriophage vectors
  • maintains foreign genes through multiple generations
  • will secrete a high yield of proteins from expressed foreign genes
iii biochemical products of recombinant dna technology
III. 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
iv genetically modified organisms gmo
IV. Genetically Modified Organisms (GMO)
  • Recombinant microbes
    • Pseudomonas syringae – prevents ice crystals
    • Bacillus thuringienisis –encodes an insecticide
  • Transgenic plants
    • Rice that makes beta-carotene
    • Tobacco resistant to herbicides
    • Peas resistant to weevils
  • Transgenic animals
    • Mouse models for CF, Alzheimer’s, sickle cell anemia
    • Sheep or goats that make medicine in their milk semen
v genetic treatments
V. Genetic Treatments
  • Gene therapy
  • Antisense DNA
  • Triplex DNA
gene therapy
Gene therapy
  • correct faulty gene in human suffering from disease
    • ex vivo – normal gene is is added to tissues taken from the body, then transfected cells are reintroduced into the body
    • in vivo – naked DNA or viral vector is directly introduced into patient’s tissue
  • Most trials target cancer, single gene defects & infections
  • Most gene deliveries are carried out by viral vectors
antisense dna targeting mrna
Antisense DNA: targeting mRNA
  • Antisense – a nucleic acid strand with a base sequence that is complementary to the translatable strand
  • Antisense DNA gets into the nucleus and binds to mRNA, blocking the expression of an unwanted protein
    • cancers
    • Alzheimer’s disease
    • autoimmune diseases
triplex dna
Triplex DNA
  • A triple helix formed when a third strand of DNA inserts into the major groove, making it inaccessible to normal transcription
  • oligonucleotides have been synthesized to form triplex DNA
    • oncogenes
    • viruses
    • receptor for IL-2
vi genome analysis
VI. Genome Analysis
  • Gene Mapping
  • DNA Fingerprinting
  • Microarray analysis
gene mapping
Gene Mapping
  • determining the location of specific genes on the chromosomes
  • Human Genome Project – to determine the nucleotide sequence of the >30,000 genes in the genome & the importance of these sequences & how they relate to human disease
dna fingerprinting
DNA Fingerprinting
  • Every individual has a unique sequence of DNA
  • Used to:
    • identify hereditary relationships
    • study inheritance of patterns of diseases
    • study human evolution
    • identify criminals or victims of disaster
microarray analysis
Microarray analysis
  • Method of determining which genes are actively transcribed in a cell under various conditions
    • health vs disease
    • growth vs differentiation
  • could improve accuracy of diagnosis and specificity of treatment