1. Topic 5: DNA Technology and Genomics
Unit 4, Topic 5 - Genetic Engineering
Topic 5: DNA Technology and Genomics

2. Unit 4, Topic 5 - Genetic Engineering
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Recombinant DNA
One purpose: To insert genes of one organism into bacteria to harvest protein
Important players:
Gene of interest (i.e. insulin, hGH, etc)
Restriction enzymes
Plasmid
Bacteria

3. Making Recombinant DNA
Unit 4, Topic 5 - Genetic Engineering
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Making Recombinant DNA
Isolate gene - Cut out the gene of interest
Cut the plasmid
Insert gene into the plasmid
Insert the plasmid into bacteria (bacterial transformation-discovered by Freddy G)
Grow bacteria and harvest the protein

4. Unit 4, Topic 5 - Genetic Engineering
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Isolating the gene
Use special proteins to cut the DNA strand at specific places
Restriction enzymes:
Target very specific base sequences
Are found in more than 100 different varieties
Are used in nature to protect bacteria from foreign invaders

5. Unit 4, Topic 5 - Genetic Engineering
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Restriction Enzymes
Each restriction enzyme recognizes a very specific nucleotide sequence
EcoR1 recognizes: GAATTC CTTAAG
The enzyme cuts it: G AATTC CTTAA G

6. Unit 4, Topic 5 - Genetic Engineering
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Unit 4, Topic 5 - Genetic Engineering

7. Unit 4, Topic 5 - Genetic Engineering
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Plasmids
Plasmids - self-replicating rings of DNA containing 2-30 genes, found in bacterial cells
Plasmid and gene cut with same restriction enzymes – Why?
Plasmid and gene have complementary “sticky” ends

8. OBJ 19

9. Unit 4, Topic 5 - Genetic Engineering
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How could you tell if the transformation really took place?
The gene of interest is attached to a promoter and some kind of marker

10. How could you tell if the transformation really took place?
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How could you tell if the transformation really took place?
Green Florescent Protein
Source bioluminescent jellyfish
Used as a visual marker of transformation

11. Insulin Production
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12. Pest-resistant crops (Bt toxin)
Unit 4, Topic 5 - Genetic Engineering
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Pest-resistant crops (Bt toxin)
Bt Corn – Transgenic corn that contains a bacterial gene for the Bt toxin
Bacillus thuringiensis – soil bacteria that produces a toxin that kills the Lepidoptera larvae

13. Gene Therapy Mutated forms of genes are replaced by functional genes.
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Gene Therapy
Mutated forms of genes are replaced by functional genes.
Currently used to treat some forms of…
Leukemia
Parkinson’s

14. Other Recombinant DNA Applications
Unit 4, Topic 5 - Genetic Engineering
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Other Recombinant DNA Applications
Herbicide-resistant crops (glyphosate)
Fast-growing fish (salmon & pout genes)
Green-glowing aquarium fish (jellyfish genes)
Spider silk expressed in
goats milk!

15. Genetic Markers Regions of DNA that vary from person to person
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Genetic Markers
Regions of DNA that vary from person to person
Used to distinguish between DNA samples
DNA Profiling (DNA fingerprinting)– identifies unique sections of DNA
Same area of DNA strand from different people (sources), different length of sequence.

16. DNA Profiling (DNA ‘fingerprinting’)
Unit 4, Topic 5 - Genetic Engineering
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DNA Profiling (DNA ‘fingerprinting’)
Purpose:
Forensic science (crime scenes)
Determining paternity
Research (disease discovery and diagnosis) – targets coding regions

17. Polymerase Chain Reaction (PCR)
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Method used to copy DNA Samples
Trace evidence can be amplified so many tests can be done
Primers – small sections of DNA
Selected to target certain DNA markers or genes

18. PCR - Steps
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Add Sample DNA, primers, DNA (Taq) polymerase to tube and free DNA nucleotide bases.
DNA polymerase from bacteria Thermus aquaticus – lives in high temperature environments
Increase sample Temperature to 94 oC
Hot enough to separate interrupt H-bonds in DNA but not degrade the strands
Reduce temperature to 54 oC – Primers base pair to open DNA Strands
Increase Heat to72 oC – Optimal functional temperature of Taq polymerase, DNA replication!!!!
Repeat Cycle about 40 times.
240 = ?????????
Thermocycler

19. Unit 4, Topic 5 - Genetic Engineering
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Gel Electrophoresis
Laboratory method for comparing different samples of DNA
Allows us to visualize DNA variations in..
Genetic markers – DNA profiling
Genes – genetic tests

20. Gel Electrophoresis Method
Unit 4, Topic 5 - Genetic Engineering
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Gel Electrophoresis Method
DNA samples are “digested” with specific restriction enzymes
Since the samples vary, the number and length of DNA fragments created will vary
Example: 2 different plasmids

21. Gel Electrophoresis Method
Unit 4, Topic 5 - Genetic Engineering
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Gel Electrophoresis Method
DNA samples are “loaded” into the “wells” of an agarose gel that is submerged in buffer
Electrical current is run through the buffer solution.
DNA is negative!! Phosphate groups PO4-
DNA fragments are pulled toward the positive pole
Smaller fragments travel farther than larger fragments

22. Unit 4, Topic 5 - Genetic Engineering
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Unit 4, Topic 5 - Genetic Engineering

23. Unit 4, Topic 5 - Genetic Engineering
Example Gel
Unit 4, Topic 5 - Genetic Engineering
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Wells
Bands of
Larger Fragments
Number of base pairs (bp)
Bands of
Smaller Fragments
DNA Ladder - DNA sample with known fragment sizes

24. Unit 4, Topic 5 - Genetic Engineering
Paternity cases
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DNA tests that targets specific regions of DNA
Genetic overlap is identified by sharing some bands with the biological Mother and Father

25. Which DNA segments are used for DNA profiling?
Unit 4, Topic 5 - Genetic Engineering
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Which DNA segments are used for DNA profiling?
Short tandem repeats (STRs) are short sequences repeated many times in a row
13 different STR sites is enough for an individual profile
PCR is used to amplify these sights and gel electrophoresis can separate the DNA segments of varying length.

26. Which DNA segments are used for DNA profiling?
Unit 4, Topic 5 - Genetic Engineering
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Which DNA segments are used for DNA profiling?
Single nucleotide polymorphisms (SNPs) are single base-pair variations (like a substitution mutation)
Restriction fragment length polymorphisms (RFLPs) are SNPs that change the length of DNA fragments created by restriction enzymes

27. Unit 4, Topic 5 - Genetic Engineering
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RFLP - Example
Restriction Fragment Length Polymorphisms (RFLPs) -

28. OBJ 24
RFLP – Example 2

30. Microarray
Method for studying expression profiles of different types of cells.
Ex. Pancreas vs. Hemoglobin
Ex. Cancer cells vs. healthy cells

32. Reproductive vs. Therapeutic Cloning
Unit 4, Topic 5 - Genetic Engineering
Reproductive vs. Therapeutic Cloning

33. Gene Therapy?

34. Unit 4, Topic 5 - Genetic Engineering
Gene Therapy
Purpose: To alter an afflicted individual’s genes and replace defective genes with normal ones

35. Unit 4, Topic 5 - Genetic Engineering

36. Unit 4, Topic 5 - Genetic Engineering
Gene Therapy
Technical issues:
Can we ensure that cells will make the right amount of the protein, at the right time, in the right place?
Can we ensure the gene’s insertion doesn’t harm the cell’s normal function?

37. Unit 4, Topic 5 - Genetic Engineering
Gene Therapy
Ethical issues:
Will it lead to eugenics, an effort to control the genetic makeup of humans?
Should we try to eliminate genetic defects in our children and their descendents?
Genetic variety is necessary for survival of a species over time!