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Met Ser A A GG AG G A G AA G Lys Glu Stop Glu Gly Val Met Stop
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Figure 2 Sequence, shown as ribose nucleotides, where genes E and D end and gene J begins. Each is out of register with the other two. The A of AUG for gene J, for example, is the second A of the UAA terminator of gene D.
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Figure 1 Presumed location of the nine genes of
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phage X174 on its circular chromosome. Transcription begins at three different places, each marked p, for promoter. The function of each gene appears within the circle.
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Figure 3 The actual map of the nine genes of phage
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X174. Note that B is entirely within A and E is entirely within D.
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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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The McGraw Hill Companies, 2001
Thirteen
Genomics, Biotechnology, and Recombinant DNA
Cloning site within lacZ gene M13 Double-stranded replicating form (RF) isolated from infected cell
MAPPING AND SEQUENCING THE HUMAN GENOME
Foreign DNA
Locating a Gene of Interest
Genes of importance can be searched for directly. A breast cancer gene provides a good example. Other genes that have been found this way include the genes for cystic brosis and Huntington disease. The concept of nding a gene is relatively simple; the methodology is tedious. Searching for many genes, including medically important genes such as one for breast cancer, means looking for a gene only by its symptoms; that is, we don t know the protein product of the gene or its location. Searching begins by looking at pedigrees of families segregating the disease and then trying to correlate the occurrence of the disease with a particular RFLP or microsatellite marker. When this is done, the gene has been localized to a particular region of a particular chromosome. Then, with a genomic library, chromosome walking (see the next section) is done until a gene in the neighborhood of the marker is found that could be the target gene. With the gene in hand, its sequence and protein product can be determined, a rst step in medical treatment.
Insert foreign DNA into cloning site; transform host E. coli for phage replication
Isolate single-stranded DNA from phage heads
3 - OH Primer
Add synthetic oligonucleotide, which hybridizes adjacent to cloning site, forming primer configuration
Chromosome Walking
Despite the limited size of any one inserted piece of foreign DNA, it is possible to learn about longer stretches of DNA by using a technique of overlapping clones called chromosome walking. Let us say that a particular gene (in region A) is located in clone 1, as discovered through probing. The cloned insert can be removed, using the same restriction enzyme initially used to insert it in the vector, and broken into small pieces that are used as probes themselves. The idea is to locate another clone with an inserted region that overlaps the rst one ( g. 13.38). The second clone is now treated the same way with segments used to probe for yet another overlap farther down the chromosome. In this way, relatively long segments of a chromosome can be available for study in overlapping clones. One obvious use of chromosome walking is to discover what genes lie next to each other on eukaryotic chromosomes.The technique is very tedious and is halted at certain areas not amenable to walking, such as repeated sequences found in the DNA of eukaryotes (see chapter 15). Once an overlapping probe contains a commonly repeated sequence, it hybridizes to many clones that do not contain adjacent segments. This cross-referencing lessens the value of the technique. Currently, newer techniques (termed chromosome jumping), designed to bypass regions not amenable to walking, are being developed. These techniques depend on the ability to locate
Figure 13.36 Phage M13, a useful vector for sequencing a
piece of cloned DNA by the dideoxy method since it exists in both single- and double-stranded forms. In addition, it contains restriction sites within a copy of the lacZ gene (blue). This allows for the selection of clones with inserted pieces of foreign DNA. An arti cial oligonucleotide, which hybridizes adjacent to the cloning site, provides the primer con guration needed for new synthesis.
Either the dideoxy or the chemical method of sequencing (not discussed) allows us to read the sequence of hundreds of nucleotides on a single gel. Whole viral, prokaryotic, and eukaryotic genomes, and numerous regions of interest in prokaryotes, eukaryotes, and viruses have been sequenced. As W. Gilbert said in his Nobel Prize acceptance speech in 1981, When we work out the structure of DNA molecules, we examine the fundamental level that underlies all processes in living cells.
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