Genetic Technologies

1. Genetic Technologies • New applications, new ethical issues

2. DNA Fingerprinting

3. DNA Fingerprinting • Used in a wide range of areas, from forensics to medicine to taxonomy, to analyze DNA. • Researchers pick out areas of interest in DNA, and often use “junk” DNA because it tends to have more mutations than genes, so has greater differences from person to person. • DNA fingerprinting can also be used to analyze genes to determine a person’s genotype for a known genetic disorder.

4. Researchers have identified short tandem repeats (STRs) that vary widely between individuals. DNA fingerprinting usually focuses on these STRs.

5. The samples are then loaded into a gel (usually agarose or polyacrylamide)

6. Different fragments have different molecular sizes, so move at different rates through the gel. Because they are polar molecules, they move in response to the electrical field across the gel.

7. At this point, the gel could be stained, photographed, and discarded. For finer analysis, such as locating specific segments of interest the gel may be placed on a nylon fiber pad, and the DNA fragments driven into the pad using a downward-directed electrical current.

8. Labeled DNA probes designed to bind to segments of interest are loaded onto the pad. These may be tagged with radioactive phosphorous or fluorescent dye.

9. Probes stick to the segments of interest, such as known STRs or specific forms of a disease-causing allele.

10. (Why do some of these people have one band, while others have two?) The resulting DNA fingerprint can then be analyzed by the experts. The segment above, for example, shows DNA from the same region for 13 different people. It could be used to determine who was at a crime scene, or identify a child’s real parents.

11. Microarrays

12. Microarrays • DNA fingerprinting is useful for testing a few genes or loci at a time. • Microarrays, however, can analyze thousands of genes, proteins, or other molecules all at once. • Microarrays are used to determine which genes in a cell are being expressed, and to analyze gene-gene interactions.

13. Microarrays • Microarrays are arrays of tens of thousands of artificial DNA probes arranged on a small glass plate. • Each probe can be designed to bind to a particular segment of interest on a particular gene or DNA locus.

14. 2. mRNA is extracted from the subject. (Why mRNA?) 1. The microarray is designed with probes that will bind to specific loci. 3. mRNA is used to synthesize cDNA, which is tagged with fluorescent dyes. 4. The cDNA is applied to the microarray and allowed to hybridize with the probes. 6. A machine scans the microarray with red and green laser light, making the probes fluoresce, and photographs the results. 5. The microarray is washed to remove cDNA that did not stick.

15. The results are analyzed to determine which genes are being expressed in specific cells, and which are turned off. The differing brightness of the fluorescence also tells researchers how much mRNA is being generated by each of the genes that is active. Researchers might, for example, compare gene expression in normal cells and cancerous cells to see which genes are active in cancer cells that are not in normal cells, and which genes are supressed in cancer cells.

16. Gene Therapy

17. Ashanti deSilva was one of the first patients to undergo gene therapy. • Ashi was born with ADA deficiency, a genetic condition in which she is missing an enzyme critical for the immune system. • Because this is a single-gene trait, it was a good candidate for gene therapy.

18. The enzyme was missing from Ashi’s white blood cells. Doctors inserted a good copy of the ADA gene into a virus known to parasitize white blood cells. • The virus successfully inserted the gene into the cells, where it began producing the enzyme.

19. However, white blood cells only live a few months. Ashi had to return several times a year for a new treatment. • The goal for ADA therapy is to treat the bone marrow stem cells that give rise to all blood cells.

20. In spite of having to return for frequent treatments, Ashi today lives a healthy, productive life. Without gene therapy, she would have no immune system and might have died of what would be a minor illness for anyone else.

21. Genetically Modified Organisms

22. Recombinant DNA • If gene therapy can “fix” genes in people, why not insert helpful genes into organisms? • Recombinant DNA technology allows researchers to take a gene from one organism and insert it into another. This has been done most successfully with plants to give them resistance to disease, pests, or herbicides.

23. Recombinant DNA is also used in “bio-pharming,” in which genes for medically therapeutic proteins are inserted into plants or into milk-producing animals. The proteins can then be purified from the plant tissue or milk for use in medical treatments. Bio-pharming may also produce fruits that produce proteins found in specific vaccines, making edible vaccines that could be grown in third-world countries. • However, altering the genes of organisms, especially those used for foods, remains highly controversial.

24. Creating a transgenic organism begins with locating the desired gene and creating a trans-gene. The DNA segment includes the desired gene and may include some “marker” gene that will be expressed in the phenotype, showing the gene has been incorporated. • Plant cells can be grown in Petri dishes, and the cells treated with the gene. • The trans gene may be inserted by a so-called “Gene Gun” that shoots small gold pellets, coated with the genes, into the cells. • Virus vectors may be used, since they already have the machinery to insert genes into a cell. • Bacteria that attack plants are also used.

25. Here, a gene is prepared for insertion into a DNA plasmid from a bacteria, which will be used to insert the gene into a plant cell.

26. The enzyme ligase seals the ends of the trans gene into the bacterial plasmid.

27. Plasmids are applied to a culture of bacteria that are known to infect plant cells.

28. This particular bacteria attacks by inserting plasmids into the plant host cell. Now it inserts the plasmid containing the trans gene.

29. If all goes well, some of the cells will incorporate the trans gene into their own DNA, where it will be expressed.

30. The transgenic cells are treated with plant hormones to grow new plants, and the plants are tested for the expression of the gene.

31. Cloning

32. Twins out of time • Lots of myths exist about cloning. Clones are not: • Mindless zombies slaves raised for organ harvest later. • Instant identical copies of yourself with all your memories. • Clones made by nuclear transfer are genetically identical to the cell donor. They are the donor’s twin, delayed by time.

33. Natural Clones • Identical twins are natural clones, created by the complete division of a fertilized egg. • Plants clone themselves when they produce shoots, runners, or other structures that take root and live independently. • Some simple animals clone themselves by budding, a form of asexual reproduction.

34. Hello, Dolly • Dolly the sheep was the first mammal produced by nuclear transfer cloning. • This process involves removing an intactcell of an adult and inserting it into an egg cell from which the nucleus has been removed. • If the egg can be stimulated to divide, it will grow into a normal embryo that can be implanted into a host animal’s uterus.

35. Dolly began life as a single cell from one breed of sheep, a white-faced Finn Dorset.

36. An egg of a Scottish Blackface ewe was harvested and its nucleus removed.

37. The two cells were stimulated with an electrical pulse to unite. This also stimulated mitosis. The egg cell carried on with multiple cell divisions as though it had been fertilized.

38. The embryo was implanted into the uterus of a Blackface ewe. Some months later, she gave birth to the white-faced lab, Dolly.

39. Cloning as a way to produce livestock is impractical. It’s far more expensive than nature’s way. • However, owners of expensive and valuable animals, such a race horses, are interested in the technique, which raises a whole new set of ethical questions. • One problem: cells seem to know how old they are. Animals born from cloned cells are born with aged cells and don’t live as long.

40. Many other genetic technologies exist, and new technologies will arise in the future. • All genetic technologies raise ethical concerns about the organisms involved and their use.