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Restriction digests can be separated by electrophoresis, then used to construct a restriction map. This is a map of the DNA showing the location of restriction enzyme recognition sites. The genetic maps, generated by mating analysis, can then be superimposed on the restriction maps, locating regions of interest on the physical map. Restriction fragment length polymorphisms (RFLPs) provide a tool for locating genes through linkage analysis and are also valuable in forensic science. The polymerase chain reaction (PCR) is a technique used to rapidly amplify particular segments of DNA.
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Tamarin: Principles of Genetics, Seventh Edition
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III. Molecular Genetics
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13. Genomics, Biotechnology, and Recombinant DNA
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STUDY OBJECTIVE 3: To study the methods of DNA se-
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DNA is usually sequenced by one of two methods. The dideoxy method developed by Sanger and his colleagues requires the synthesis of DNA in the presence of chainterminating (dideoxy) nucleotides. Electrophoresis followed by autoradiography allows the sequence of nucleotides synthesized to be determined directly. Fluorescent labeling allows computerized sequence determinations. The phage X174 was sequenced in its entirety through the forerunner of this technique, the plus-andminus method. Gilbert and Maxam s chemical method also is used widely. STUDY OBJECTIVE 4: To look at the goals and methods of
genome. Initial success was announced in the spring of 2000. Modern linkage maps are being created of restriction sites, microsatellite markers, sequence-tagged sites, and single-nucleotide polymorphisms. They are being coordinated with physical maps created with overlapping contiguous clones of chromosomes. These techniques currently allow us to nd genes of interest. The project also includes the sequencing of the genomes of other relevant organisms. STUDY OBJECTIVE 5: To look at the practical bene ts and
human issues of genetic engineering 397 398
Genetic engineering is moving forward on a number of fronts. Medical, agricultural, and industrial applications are becoming widespread.
the Human Genome Project
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The Human Genome Project is a massive, international effort to map and sequence all 3.3 billion bases of the human
PROBLEM 1: A piece of eukaryotic DNA is obtained by
when a desired event has taken place. In this chapter, we discussed the re y luciferase reporter system in which the desired result (transcription of a particular promoter) causes a transgenic tobacco plant to glow. Let us say that we are studying the control of transcription of a particular eukaryotic gene. We could attach the promoter of that gene to the re y luciferase gene in a Ti plasmid by cloning techniques. The plasmid could then transfect tobacco plants, and we could continue our experiment to determine whether the promoter under study is active under various conditions. We would know whether it was active by watering the plants with luciferin. If the plant glows, then the luciferase gene product is present, which means that the promoter under question is active. In other words, the glowing of the plant reports the action of the promoter under question; the promoter is active because it allowed the transcription of the luciferase gene. We also discussed the green uorescent protein reporter system.
PROBLEM 3: A piece of DNA has the sequence 3 -GGCG-
using a restriction endonuclease that leaves blunt ends (HaeIII). How could we get this piece of DNA into a BamHI site in plasmid pBR322, and how would we know when the foreign DNA has been cloned Answer: Since the two pieces of DNA (the eukaryotic piece and the plasmid) have different ends, they must be made compatible before cloning. The simplest way would be to attach blunt-ended linkers to the foreign DNA with phage T4 DNA ligase (see g. 13.9). The linkers, of course, would have a BamHI site within. After the linkers are attached to the foreign DNA, it would be treated with the BamHI restriction enzyme, giving the foreign DNA BamHI ends. The plasmid is then also treated with the restriction enzyme and the two (the foreign DNA and the cut plasmid) are now mixed together in the presence of E. coli DNA ligase, which seals up the plasmids, with or without cloned inserts (see g. 13.6). Since they have compatible ends, some of the time, a piece of foreign DNA is inserted into a plasmid. The plasmids are then taken up by E. coli cells that are grown overnight in an incubator.The bacterial colonies are then replica-plated on media with the antibiotics ampicillin or tetracycline. Colonies that are resistant to ampicillin but sensitive to tetracycline are assumed to be bacteria containing plasmids with cloned inserts (see g. 13.8).
PROBLEM 2: How does a reporter system work Answer: A reporter system is a genetically manipulated system that displays a particular phenotype or reaction
TATTC-5 . It is sequenced using the dideoxy method. How many bands are found on the ladder gel How many bands and of what size are found for each reaction mixture Answer: Since the piece of DNA is nine bases long, the total number of bands in all four lanes of a sequencing gel add up to nine (see g. 13.33). By each reaction mixture, we mean the four reaction mixtures each with one of the dideoxynucleotides. In the reaction mixture with ddTTP,
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