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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
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Mapping and Sequencing the Human Genome
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Figure 13.37 Processed data from automated DNA analysis using uorescent dyes. The DNA is
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sequenced by attaching a different uorescent dye to each dideoxy base. Thus, the dideoxy bases can be identi ed by their uorescent color in a laser light rather than by which lane they occupy in a gel. Only one lane need therefore be run. In this diagram, guanine is yellow, cytosine is blue, adenine is green, and thymine is red. The sequence is read left to right, top to bottom. (From L. Johnston-Dow,
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et al., BioTechniques, 5:754 65, 1987, copyright 1987. Eaton Publishing, Natick, MA. Reprinted with permission.)
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the two ends of a segment without having to walk through the middle. Ends of a segment can be located if the region has been inverted or if a large region is cloned and the middle part later removed, leaving just the ends. A probe of the ends allows the investigator to locate clones with rst one end and then the other, effectively jumping over the intervening region.
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The Breast Cancer Gene
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The initial location of the breast cancer gene BRCA1 was determined by M. King in 1990 using a marker (D17S74) on the long arm of chromosome 17 ( g. 13.39); it was the 183rd marker that King had tried ( g. 13.40). The breast cancer gene BRCA1 was particularly dif cult to locate because it accounts for only about 5% of all breast canMary-Claire King (1946 ). (Courtesy of
Of ce of Public Information, Berkeley Campus, University of California. Photograph Jane Scherr.)
cers. However, it accounts for a much higher percentage of inherited, early onset breast cancers, those in women under fty years of age. One woman in two hundred inherits this gene, and among those women, 80 to 90% risk developing the disease. The actual locating and cloning of this gene was done in 1994 by a team led by M. Skolnick. The gene codes for a protein of 1,863 amino acids; it seems to act as a tumor suppressor protein (see chapter 16). Its mechanism of action is as a transcription factor associated with RNA polymerase II (see chapter 10).
The Human Genome Project
The Standard Method
In chapter 6, we developed a human chromosome map. Generally, a locus was located on a particular chromosome by tissue culture techniques (somatic-cell hybridization). Loci could be pinpointed further using aberrant chromosomes, such as those with deletions. If a locus was present when the intact human chromosome was present but absent if the deletion chromosome was present, the gene could be localized to the deleted region. In addition, probes for speci c genes can show us roughly where that gene is located ( g. 13.41).
Tamarin: Principles of Genetics, Seventh Edition
III. Molecular Genetics
13. Genomics, Biotechnology, and Recombinant DNA
The McGraw Hill Companies, 2001
Thirteen
Genomics, Biotechnology, and Recombinant DNA
Two general methods were developed for mapping the human genome, the standard method, supported in large part by federal funding, and the whole-genome shotgun method used by the Celera Genomics Company. In the standard method, the project is reduced to nding a segment of the genome and locating where it belongs. The segment is then sequenced. By the overlap of sequenced pieces, the whole genome is pieced together. Mapping is done chromosome by chromosome since individual chromosomes can be isolated in large numbers by the methods of ow cytometry, described in chapter 15. In the initial stages of the Human Genome Project, when the primary task was mapping, yeast arti cial chromosomes (YACs) were the primary cloning agent. However, as the emphasis of the project shifted
Figure 13.38 Chromosome walking technique. This technique allows one to study long chromosomal regions by locating overlapping cloned inserts. We begin with a speci c cloned piece of DNA, referred to as insert A. This piece is fragmented to create probes for other clones in a genomic library that contains regions that overlap A (the next region down is referred to as B). The A-B clone is itself then fragmented to create probes to repeat the process, moving down the chromosome.
13 12 p 11.2 11.1 11.1 11.2 12 21.1 21.2 21.3 22 23 24 25 Chromosome 17 Figure 13.39 A Giemsa-banded chromosome 17, showing the Figure 13.40 A pedigree of a family of individuals in which
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