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2 b. Genetic engineering techniques can manipulate the heritable information of DNA and, in special cases, RNA. To demonstrate student understanding of this concept, make sure you can explain: Electrophoresis Plasmid-based transformation Restriction enzyme analysis of DNA Polymerase Chain Reaction (PCR)

3 2. Restriction enzymes are used to make recombinant DNA Gene cloning was made possible by the discovery of restriction enzymes that cut DNA molecules at specific locations. Bacteria use restriction enzymes to cut foreign DNA, such as from phages or other bacteria. Restriction enzymes are very specific, recognizing short DNA nucleotide sequences and cutting at specific points in these sequences. Bacteria protect their own DNA by methylation.

4 Each restriction enzyme cleaves a specific sequence of bases or restriction site. These are often a symmetrical series of four to eight bases on both strands running in opposite directions. If the restriction site on one strand is 3 -CTTAAG-5, the complementary strand is 5 -GAATTC-3. Notice at either end there are compliments, then the next ones in are compliments, etc. These are called inverted repeats. Identical copies of a DNA molecule will always yield the same set of restriction fragments when exposed to a specific enzyme.

5 Restriction enzymes cut bonds of both strands, often in a staggered way, creating single-stranded ends, sticky ends on each cut piece (each piece is called a restriction fragment). These sticky ends will form hydrogen-bonds with complementary single-stranded stretches on other DNA molecules cut with the same restriction enzyme. Why does it need to be the same???

6 Restriction enzymes and DNA ligase can be used to make recombinant DNA, DNA that has been spliced together from two different sources. Watch Restriction Endonucleases and Early Genetic Engineering Experiments here. Fig. 20.2

7 Common test question: Practice figuring out what size fragments would be produced by a specific enzyme, given the DNA, some markers which tell you what length it is, and places where the enzyme cuts (recognition sequences). It is really just simple addition and subtraction math. You don t want to miss these simple ones. Let s practice. (4:23) And here is another. (4:04) Now a contest from Plasmid Mapping Exercises

8 3. Genes can be cloned with the help of bacteria cells and their plasmids Several methods can get a gene into a cell. If some object (like a virus) acts like a delivery truck, we call that object a VECTOR. Let s look at how plasmids can be used as vectors to get a gene into a bacteria cell.

9 b. Genetic engineering techniques can manipulate the heritable information of DNA and, in special cases, RNA. To demonstrate student understanding of this concept, make sure you can explain: Electrophoresis Plasmid-based transformation Restriction enzyme analysis of DNA Polymerase Chain Reaction (PCR)

10 Let s review with pictures. The process of cloning a human gene in a bacterial plasmid can be divided into five steps. udent_view0/chapter16/anim ations.html# See: Steps in Cloning a Gene Fig. 20.3

11 5. Identifying cell clones with the right gene. 6. Let s see how nucleic acid probes are used to find the bacteria cell that took up the gene you want. A radioactive isotope or fluorescent protein labels the probe.

12 Fig. 20.4

13 Green flourescent protein is a cool, Nobel Prize winning story. Watch here to see its discovery. 3 min. See the structure here. And here to see a neat new application. 16 min.

14 As we have seen, we also use this technology to let bacteria make proteins we can use. Transformation is a simple way to get a gene into bacteria cells. These include human insulin (the first one made) and growth factor (HFG). Human insulin, produced by bacteria, is superior for the control of diabetes than the older treatment of pig or cattle insulin. Human growth hormone benefits children with hypopituitarism, a form of dwarfism. Today, however, it is highly mis-used as an anti-aging drug.

15 But what about that intron/exon thing? If you just want to clone the gene to get several copies from these bacteria, no problem, but If you want this bacteria cell to make proteins from this eukaryotic gene, you have a problem It doesn t have spliceosomes to cut out introns, sooo Make some cdna Using the mrna s in the eukaryotic cell s cytoplasm, which have already been edited, use it to make complementary DNA (cdna), then put that into the plasmid. Voila! Watch cdna here.

16 Other ways to clone a gene BAC s Bacterial Artificial Chromosomes, and YAC s Yeast Artificial Chromosomes Advantage they can hold a bigger set of genes YAC s will also have eukaryotic promotors and enhancers, and can cut out introns. The cutting and pasting process is basically the same.

17 5. The polymerase chain reaction (PCR) clones DNA entirely in vitro DNA cloning is the best method for preparing large quantities of a particular gene or other DNA sequence. When the source of DNA is scanty or impure, the polymerase chain reaction (PCR) is quicker and more selective, but can only copy small pieces. This technique can quickly amplify any small piece of DNA without using cells. Watch this animations.html#

18 b. Genetic engineering techniques can manipulate the heritable information of DNA and, in special cases, RNA. To demonstrate student understanding of this concept, make sure you can explain: Electrophoresis Plasmid-based transformation Restriction enzyme analysis of DNA Polymerase Chain Reaction (PCR)

19 The DNA is incubated in a test tube with special DNA polymerase, a supply of nucleotides, and short pieces of single-stranded DNA as a primer. Fig. 20.7

20 In PCR, a three-step cycle: heating, cooling, and replication, repeated many times, brings about a chain reaction that produces an exponentially growing population of DNA molecules. The key to easy PCR automation was the discovery of an unusual DNA polymerase (Taq polymerase), isolated from bacteria living in hot springs, which can withstand the heat needed to separate DNA strands at the start of each cycle. Note that probes and primers are pretty similar both are short strands of nucleotides with bases complimentary to those of another strand of interest. But help start replication, and radioactive probes help find the piece of DNA you want.

21 Devised in 1985 by Kerry Mullis, PCR has had a major impact on biological research and technology. He even has a song about it. 2 min. PCR has amplified DNA from a variety of sources: fragments of ancient DNA from a 40,000-year-old frozen wooly mammoth, DNA from tiny amounts of blood or semen found at the scenes of violent crimes, DNA from single embryonic cells for rapid prenatal diagnosis of genetic disorders, DNA of viral genes from cells infected with difficult-todetect viruses such as HIV. Not another song! 3 min.

22 Introduction Once we have prepared samples of DNA, each containing a large number of identical segments, we can begin to ask some far-ranging questions. These include: Are there differences in a gene in different organisms? Where and when is a gene expressed? What is the location of a gene in the genome? How has a gene evolved, as revealed by interspecific comparisons?

23 You can use this to know the nucleotide sequence of the gene and ultimately the sequences of entire genomes. One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis. Gel electrophoresis separates macromolecules - nucleic acids or proteins - on the basis of their rate of movement through a gel in an electrical field. Rate of movement depends on size, electrical charge, and other physical properties of the macromolecules, just like what other separation technique?

24 Separation depends mainly on size, shorter pieces move more easily through the gel, therefore travel further than larger pieces. Fig. 20.8

25 1. Restriction fragment analysis detects DNA differences that affect restriction sites After cutting DNA molecules with a restriction enzyme, the fragments can be separated by size via gel electrophoresis. This produces a pattern of bands. The separated fragments can also be recovered undamaged from gels, providing pure samples of individual fragments. Watch here first.

26 We can use nucleic acid hybridization with a specific probe to label discrete bands that contain our gene of interest. The radioactive label on the single-stranded probe can be detected. Watch Southern Blot here.

27 This so-called DNA profiling can be used to identify the criminal or father in a paternity suit. We start by adding the restriction enzyme to each of the three samples to produce restriction fragments. We then separate the fragments by gel electrophoresis.

28 For our three individuals, the results of these steps show that individual III has a different restriction pattern than individuals I or II. This is a common, simple test question. Fig

29

30 Southern blotting can be used to examine differences in noncoding DNA as well. Remember those repeats (satellite DNA) we all have different numbers of copies of? These restriction fragment length polymorphisms (RFLPs) can serve as a genetic marker for a particular location (locus) in the genome. Let s practice.

31 Time now for a virtual lab

32 Here is a specific example a famous chapter in biology. If we have time, let s look at this video about Huntington s Disease. If we have time, how about a Socratic seminar?

33 One ambitious research project made possible by DNA technology has been the Human Genome Project, begun in This is an effort to map the entire human genome, and determine the complete nucleotide sequence of each human chromosome.

34 Let s see how sequencing works Link to How To Sequence a Genome and go through all the steps. Combined with your knowledge of how electrophoresis works, it is really neat to see how the sequencer contraption works. But this newer method is even neater. 4:05

35 3. Genome sequences provide clues to important biological questions Here are three basic outcomes of the project: Better understand diseases Develop gene libraries Produce gene probes Here is how a thing called a microchip is used to diagnose which virus might be infecting someone. (2:10) And here s the Chilean Blob Story, if time.

36 GENE THERAPY Techniques for gene manipulation hold great potential for treating disease by gene therapy. This alters an afflicted individual s genes. A normal allele is inserted into somatic cells of a tissue affected by a genetic disorder. For gene therapy of somatic cells to be permanent, the cells that receive the normal allele must be ones that multiply throughout the patient s life. RNAi is one type of gene therapy. Remember it?

37 Bone marrow cells, which include the stem cells that give rise to blood and immune system cells, are prime candidates for gene therapy. A normal allele could be inserted by a viral vector into some bone marrow cells removed from the patient. If the procedure succeeds, the returned modified cells will multiply throughout the patient s life and express the normal gene, providing missing proteins. Fig

38 Retroviruses, as we have already seen, can deliver genes into cells Cystic Fibrosis was one of the first diseases treated by gene therapy. Engineered retroviruses delivered via an inhaler delivered a healthy chlorine transport protein gene to cells lining the upper respiratory tract. Here s a Parkinson s Disease example. 2:41 Or watch here to see liposomes as a vector. :31 Another nano-robots, vector of the future? 2:19

39 And how about this way to change genes? Did you know you have zinc fingers? TALENS? Watch here. 3:37 The zinc fingers cut genes. When they are repaired the gene is often disrupted, or if cuts are made and a new gene delivered to the cell at the same time the new gene may be inserted into the chromosome.

40 Gene therapy vs. GMO s While most people see the value in using DNA technology for medical applications, many draw the line with the intentional modification of organisms to give them desired traits. Not all gene modification is for therapy, either, so All gene therapy involves gene modification, but not all gene modification is done for therapy (trying to fix something that is wrong).

41 Discuss the ethical and legal considerations that the biotechnology revolution has generated. Provide multiple real-life examples of these issues. Offer multiple lines of evidence to support and refute these considerations.

42 3. DNA technology raises important safety and ethical questions The power of DNA technology has led to worries about potential dangers. For example, recombinant DNA technology may create hazardous new pathogens. In response, scientists developed a set of guidelines that in the United States and some other countries have become formal government regulations. Now, let s look at one of the ethical aspects. 8:30

43 c. Illustrative examples of products of genetic engineering include: Genetically modified foods Transgenic animals Cloned animals Pharmaceuticals, such as human insulin or factor X

44 Today, most public concern centers on genetically modified organisms (GMO s) used in agriculture. GMO s have acquired one or more genes (perhaps from another species) by artificial means. Genetically modified animals are still not part of our food supply, but GM crop plants are.

45 Transgenic organisms are GMO s with genes from another species so they can exploit the attributes of the new genes (for example, faster growth, larger muscles). Some crops are made resistant to cold or salt. Other transgenic organisms are pharmaceutical factories - a producer of large amounts of an otherwise rare substance such as the clotting Factor IX produced in sheep milk Fig

46 In particular, transgenic plants may pass their new genes to close relatives in nearby wild areas through pollen transfer. Transference of genes for resistance to herbicides, diseases, or insect pests may lead to the development of wild superweeds that would be difficult to control.

47

48 Agricultural scientists have engineered a number of crop plants with genes for desirable traits. These includes delayed ripening tomatoes (Flavr saver) and resistance to insects and disease. The Ti plasmid, from the soil bacterium Agrobacterium tumefaciens, is often used to introduce new genes into plant cells. The Ti plasmid normally integrates a segment of its DNA into its host plant and induces tumors.

49 The recombinant plasmid can be put back into Agrobacterium, which then infects plant cells, or introduced directly into plant cells. Fig

50 Scientists are using gene transfer to improve the nutritional value of crop plants. For example, a transgenic rice plant has been developed that produces yellow grains containing beta-carotene. Humans use beta-carotene to make vitamin A. Currently, 70% of children under the age of 5 in Southeast Asia are in vitamin A, leading to vision impairment and increased disease rates. deficient Fig

51 Here are some ways other than transformation to get DNA into a cell: In electroporation, electrical pulses create a temporary hole in the plasma membrane through which DNA can enter. Alternatively, scientists can inject DNA into individual cells using microscopically thin needles. In a technique used primarily for plants, DNA is attached to microscopic metal particles and fired into cells with what is called a gene gun.

52 To develop a transgenic animal, scientists remove ova from a female and fertilize them in vitro. The desired gene from another organism is inserted into the nuclei of the eggs. The engineered eggs are then surgically implanted in a surrogate mother. If development is successful, the result is a transgenic animal, containing genes from a third parent, even from another species. Do your pets light up when you come home? 1:00

53 Increasingly, genetic engineering is being applied to environmental work. For example genetically engineered microbes that can extract heavy metals from their environments and incorporate the metals into recoverable compounds may become important both in mining materials and cleaning up toxic mining wastes. Oil-eating microbes can help clean up oil spills.

54 CLONING (not genes this time!): Making animals or controversial embryos Dolly the sheep was the first clone of an adult mammal produced by the technique known as SCNT, or somatic cell nuclear transfer 2:00 (see next page). This is called reproductive cloning. Other cloning had been done for years by separating embryo cells. Clones are exact genetic copies. Eg: identical twins. Plant clones are easy, as are some animals, eg; jellyfish, starfish

55 Reproductive cloning. Nuclear transfer technique- for cloning more complex animals. Nucleus of all cells contain complete DNA. Nucleus of egg cell is removed/destroyed Replaced by nucleus from somatic cell Result is as if the egg had been fertilized. It has the diploid # of chromosomes, and can develop into an exact copy of whoever s nucleus was transplanted. Advantage- exactly reproduce individuals with desired traits. NOTE how this is similar yet different to how GMO animals were produced. In GMO s, an egg kept it s nucleus, had a gene put in it, and then was fertilized by a sperm in vitro. The GMO had 2 parents was not a clone.

56 That was REPRODUCTIVE cloning you want to make a copy of an organism because you really valued it. Contrast that with THERAPEUTIC cloning. Cloning an embryo to use its cells as embryonic stem cells (now federally funded in the U.S.) is called therapeutic cloning. Watch. Watch. Watch. 2 min. each. Both have big moral and legal issues. See NOVA stem cells segment on DVD. (about 8:15) And watch an example of the latest hope. (green mice) 1:49 Then, if time, deer, salamander and zebra fish animations from Potent Biology DVD (1 min. each)

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