1. Manipulating DNA

2. Selective Breeding
Man has selectively bred plants and animals for desired traits

3. Mutations of Pointsettias induced by radiation

4. New DNA Technology
Now DNA can be manipulated to produce new combinations not found in nature
How far should we go?
We need to set bioethical limits

5. Restriction Endonucleases
In 1980, geneticists spliced the human gene that encodes interferon into a bacterial cell’s genome.
Marked beginning of genetic engineering.
Through natural selection, some bacteria contain enzymes, restriction endonucleases, that fragment viral DNA.
Recognize specific nucleotide sequences, and cleave the DNA.
They recognize palindromes
Methylate their own DNA for protection

6. Restriction Endonucleases
Sequences recognized by restriction endonucleases are typically four to six nucleotides long, and are often palindromes.
Restriction enzymes can bind to and cleave both strands of DNA.
Raven - Johnson - Biology: 6th Ed. - All Rights Reserved - McGraw Hill Companies

7. Restriction Endonucleases
There are hundreds of bacterial restriction endonucleases, each with a specific recognition sequence.
Given restriction endonuclease can cut DNA from any source into fragments.
Complementarity, single-stranded ends can pair with each other.
Sticky Ends
Fragments joined together with DNA ligase.

8. Using RE’s to Manipulate Genes
Chimeras
One of first recombinant genomes produced by genetic engineering was a bacterial plasmid into which an amphibian ribosomal RNA gene was inserted.
Viruses can also be used as vectors to insert foreign DNA into host cells.

10. Genetic Engineering Experiment
Four Stages
DNA Cleavage
Restriction endonuclease cleaves source DNA into fragments.
Production of Recombinant DNA
DNA fragments inserted into plasmids or viral vectors.
Cloning
Screening

11. Genetic Engineering Experiment
Screening
Clones with DNA fragment of interest are identified from the clone library.
Preliminary Screening - Eliminate any clones without a vector and clones with vectors that do not contain DNA.
Employ vector with gene for antibiotic resistance and lac Z’ gene.
Expose to growth medium.

12. Genetic Engineering Experiment
Secondary Screening (Gene of Interest)
Hybridization - Cloned genes form base pairs with complementary sequences on another nucleic acid (probe).
Grow on agar and then transfer to a filter pressed on colonies.
Treat filter with radioactive probe, and perform autoradiography.

13. Working With Gene Clones
Polymerase Chain Reaction (PCR)
Used to copy specific gene sequences.
Three Basic Steps
Denaturation
Annealing of Primers
Primer Extension

14. Restriction Fragment Length Polymorphism (RFLP) Analysis
DNA is cut into small pieces using restriction enzymes
Many copies are made using PCR
Radioactive probes attached to mark ends
An electric current is used to separate pieces by size in a gel
Larger pieces are slower and don’t move as far down the gel

15. DNA Fingerprinting DNA is unique for every individual
Comparing the sequences of small pieces of DNA can be used:
Find relationships
Narrow crime suspects
Identify organisms

16. DNA Sequence Technology
Genome Sequencing
Due to technological advances, sequencing of entire genomes is now practical.

17. DNA Sequence Technology
Human Genome Project
Human genome contains around 3.2 billion bases and about 30,000 genes.
Human genes are quite fragmented.
Fully one-third of genome is composed of noncoding repeated sequences.
Genes are not evenly distributed over the genome.

18. DNA Sequence Technology
Proteonomics
Cataloging and analyzing every protein in the human body.
Each gene’s nucleotide sequence specifies an amino acid sequence folded in a certain way.

19. Biochips
Gene Microarrays (Biochips) are small squares of glass covered with millions of strands of DNA.
Scanning beam used to build nucleotides.
Look at Single Nucleotide Polymorphisms (SNP’s).
Cancer Screening
Personal Privacy Issues

21. Medical Applications Pharmaceuticals
Introduction of protein-encoding genes.
Atrial peptides - High blood pressure and kidney failure.
Tissue plasminogen activator - Dissolving blood clots.

22. Medical Applications Gene Therapy
Add working copies of single defective gene.
Leber's congenital amaurosis
X-linked SCID
Adrenoleukodystrophy
chronic lymphocytic leukemia
acute lymphocytic leukemia
multiple myeloma
Parkinson's disease
These recent clinical successes have led to a renewed interest in gene therapy

23. Medical Applications Piggyback Vaccines
Produce subunit vaccines against viruses.
Herpes
Hepatitis
DNA Vaccine
Cellular immune response.

25. Agricultural Applications
Ti plasmid has been early successful vector.
Herbicide Resistance
Insert genes encoding for proteins making crops resistant to herbicide.
Widespread herbicide use possible.
Nitrogen Fixation
Introduce genes that allow crops to fix nitrogen.
Reduce need for fertilizer.

26. Agricultural Applications
Insect Resistance
Insert genes encoding proteins harmful to insects.
Real Promise - Produce genetically modified plants with traits benefiting consumers.
Iron Deficiency in developing countries
Transgenic Rice
Increasing milk production
Bovine Somatotrophin

28. Cloning
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Raven - Johnson - Biology: 6th Ed. - All Rights Reserved - McGraw Hill Companies

29. Tools needed in Gene Cloning
Restriction enzymes
Cuts the desired DNA strands
Bacterial plasmid
cloning vector used to move DNA to another organism
Host DNA
Donor gene(s)
Produces a transgenic organism with recombinant DNA

30. Clones
Copies of one organism

31. Clones Produced by taking cuttings from one plant Grafting Budding
Rooted cuttings

32. Clones
Produced for many years by dividing an embryo to form identical quadruplets
Cows
Pigs

33. A New Type of Clone The nucleus of a donor cell
The ova (egg) of another organism
A surrogate mother

34. How it’s done…

35. FDA says cloned animals safe to eat
Food producers, including Tyson Foods Inc. and Dean Foods Co., said they won't use cloned foods anytime soon.
Bloomberg News / January 16, 2008
WASHINGTON - Cloned cows, pigs, goats, and their offspring are safe to enter the US food supply, regulators said over protests from lawmakers, consumer groups, and worried eaters.

36. Maryann Mott for National Geographic News
April 4, 2006
Meet Royal Blue Boon Too, the first commercially cloned horse in the United States.
The cost of the frisky foal: U.S. $150,000.
That may be a bargain, considering that the original Royal Blue Boon, a 26-year-old American Quarter Horse now past breeding age, has earned more than $380,000 as a competition and show horse. .
Champion Horse Cloned by Texas Breeder

37. Some problems to Overcome:
Many casualties
Birth defects
Failures
Premature aging
Improve technique

38. Stem Cells Embryonic Stem Cells Tissue Specific Stem Cells
Cells produced very early in development- each capable of developing into a healthy individual.
Can develop into any tissue.
May provide ability to restore damaged tissue.
Tissue Specific Stem Cells
As development proceeds, tissue-specific stem cells persist.

39. Controversial topics…
Scientists hope to use cloned embryos to harvest precious "stem cells" — cells that, in theory, can ultimately grow into any kind of cell in the body.
These cells could be used to generate new organs or cell clusters to treat patients with failing organs or degenerative diseases
In the case of transplant patients, organs made from exact copies of an individual's own cells and would not be rejected.
This research is still in its earliest phase of development. All of the embryos generated actually died — none grew beyond the six-cell stage
Controversial: The thinking is that by allowing any kind of cloning of human embryos, it is then not such an enormous leap to apply the same techniques to making copies of people.

40. Ethics and Regulation Questions
How do we measure the Potential Risks of Genetically-Modified Crops?
Is Eating Genetically Modified Food Dangerous?
Are Genetically Modified Crops harmful to the Environment?
Should We Label Genetically Modified Foods?

41. Copyright © McGraw-Hill Companies Permission required for reproduction or display