Copyright 2002 Pearson Education, Inc., publishing as Benjamin Cummings

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2 Here s one thing genetic engineers do: Techniques for gene cloning enable scientists to prepare multiple identical copies of gene-sized pieces of DNA. Cloning means to make copies, in this case, copies of genes. We also use the word to describe making copies of cells or organisms. Did you know you can now have your dog cloned?

3 2. Restriction enzymes are used to make recombinant DNA Genetic engineering was made possible by the discovery of restriction enzymes that cut DNA molecules at specific locations. In nature, bacteria use restriction enzymes as a defense chemical to cut foreign DNA, such as from viruses or other bacteria. Most restrictions enzymes are very specific, recognizing short DNA nucleotide sequences and cutting at specific points in these sequences. Let s watch Choose Restriction Endonucleases.

4 Restriction enzymes cut DNA often in a staggered way, creating single-stranded ends called sticky ends on each cut piece. These sticky ends will form hydrogen-bonds with complementary single-stranded stretches on other DNA molecules cut with the same restriction enzyme. This allows any two pieces of DNA from any two organisms, like you and your dog, to be combined together, which is one of the big areas of genetic engineering.

5 Here s a diagram of how restriction enzymes can be used to make recombinant DNA, DNA that has been spliced together from two different sources. Watch open the folder, then the MP4 file. Fig. 20.2

6 3. Genes can be cloned with the help of bacteria cells and their plasmids Recombinant plasmids are produced by splicing restriction fragments from foreign DNA into plasmids. Know what plasmids are? These can be returned by transformation to bacteria cells (different bacteria cells than the ones who gave them up). Then, as a bacterium carrying a recombinant plasmid reproduces, the plasmid replicates within it. Voila! Clones!!

7 Let s review with pictures. The process of cloning a human gene in a bacterial plasmid can be divided into five steps. Now with an animation. Fig. 20.3

8 Just let them reproduce and you have tons of bacteria with your selected human gene in it. One thing you can do is let them make lots of the human protein. These include human insulin and growth factor (HFG). What would be the advantages over getting these chemicals from a natural source?

9 Now that we have lots of pieces of DNA, here s another thing we can do. 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 that you did with plant pigments?

10 For linear DNA molecules, separation depends mainly on size (length of fragment), shorter pieces move more easily through the gel, therefore travel further than larger pieces. Watch open folder & MP4 Fig. 20.8

11 So-called DNA profiling can be used to identify the criminal or father in a paternity suit because the pattern of a person s bands from electrophoresis of their DNA is like a supermarket bar code, it is unique. So if the criminal left some of their DNA at the crime scene, here s what you do: watch first the one on Rest. Frag. Length Polymorphisms We start by adding the same restriction enzyme to each of the three samples to produce restriction fragments. We then separate the fragments by gel electrophoresis.

12 For our three individuals, the results of these steps show that individual III has a different restriction pattern than individuals I or II. Shall we have a Clue?? Fig

13 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, ultimately by determining the complete nucleotide sequence of each human chromosome. An international, publicly funded consortium has proceeded in three phases: genetic (linkage) mapping, physical mapping, and DNA sequencing. In addition to mapping human DNA, the genomes of other organisms important to biological research are also being mapped. These include E. coli, yeast, fruit fly, and mice.

14 The surprising - and humbling - result to date from the Human Genome Project is the small number of human genes, 20,000 to 22,000. This is far less than expected and only two to three times the number of genes in the fruit fly or nematodes. Humans have enormous amounts of DNA that doesn t for proteins. code

15 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 in a similar way to how a gene was put into a bacteria cell. 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.

16 Bone marrow cells, which include 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 virus 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

17 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 highly toxic mining wastes.

18 Transgenic organisms is this Frankensteinish?? Some crops are made resistant to cold or salt. Down on the pharm, these sheep have some special milk. Fig

19 To develop a transgenic organism, scientists remove ova from a female and fertilize them in vitro. The desired gene from another organism is cloned and then inserted into the nuclei of the eggs. Some cells will integrate the foreign DNA into their genomes and are able to express its protein. 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.

20 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 deficient in vitamin A, leading to vision impairment and increased disease rates. Fig

21 Today, most public concern centers on genetically modified organisms (GMO s) used in agriculture.

22 So what s up with this GMO thing? Well, let s watch a couple of videos. First Second Shall we test what you eat?

23 As with all new technologies, developments in DNA technology have ethical overtones. Who should have the right to examine someone else s genes? How should that information be used? Should a person s genome be a factor in suitability for a job or eligibility for life insurance? The power of DNA technology and genetic engineering demands that we proceed with humility and caution.

24 Cloning on a large scale: Making sheep or controversial embryos Dolly the sheep was the first clone of an adult mammal produced by the technique known as nuclear transfer. This is called reproductive cloning. Watch here Cloning an embryo to use its cells as embryonic stem cells (not federally funded in the U.S. right now) is called therapeutic cloning. Both have big moral and legal issues.