The document provides an overview of genetic engineering and biotechnology tools and techniques, including: - Restriction enzymes, ligase, and reverse transcriptase which are used to cut, splice and reverse DNA/RNA - Methods for analyzing DNA like gel electrophoresis, hybridization probes, sequencing, and PCR - Cloning vectors like plasmids and cloning hosts like E. coli that are used to replicate and express recombinant DNA - Applications of recombinant DNA technology like producing insulin, vaccines, and transgenic organisms - Genetic engineering techniques like gene therapy, antisense DNA, and triplex DNA for treating diseases - Genome analysis methods like gene mapping, DNA fingerprinting and microarray analysis.

1. PowerPoint to accompany
Foundations in Microbiology
Fifth Edition
Talaro
Chapter
10
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

2. Genetic Engineering: A
Revolution in Molecular Biology
Chapter 10 2

3. Genetic engineering
• direct, deliberate modification of an
organism’s genome
• bioengineering
• Biotechnology – use of an organism’s
biochemical and metabolic pathways for
industrial production
3

4. I. Tools & Techniques of genetic
engineering
• enzymes for dicing, splicing, & reversing
nucleic acids
• analysis of DNA
4

5. 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
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6. 6

7. 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
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8. Gel electrophoresis
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9. Southern blot hydridization
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10. In situ hybridization
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11. Sanger DNA sequence technique
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12. Polymerase chain reaction (PCR)
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13. II. Methods in Recombinant
DNA Technology
• concerned with transferring DNA from
one organism to another
3. Cloning vectors & hosts
4. Construction of a recombinant plasmid
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14. 14

15. 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
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16. Vector considerations
• origin of replication
• size of donated DNA vector will accept
• gene which confers drug resistance to their
cloning host
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17. pBR322
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18. Characteristics of cloning hosts
1. rapid overturn, fast growth rate
2. can be grown in large quantities using ordinary culture
methods
3. nonpathogenic
4. genome that is well delineated
5. capable of accepting plasmid or bacteriophage vectors
6. maintains foreign genes through multiple generations
7. will secrete a high yield of proteins from expressed
foreign genes
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19. 19

20. 20

21. 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
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22. 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
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23. Bioengineering of plants
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24. Transgenic mice
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25. V. Genetic Treatments
• Gene therapy
• Antisense DNA
• Triplex DNA
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26. 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
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27. Gene therapy
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28. 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
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29. 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
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30. Antisense DNA & triplex DNA
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31. VI. Genome Analysis
• Gene Mapping
• DNA Fingerprinting
• Microarray analysis
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32. 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
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33. Map of chromosome 16
33

34. 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
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35. DNA fingerprints
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36. Pedigree analysis
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37. 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
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38. Microarray
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