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Tamarin: Principles of Genetics, Seventh Edition
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13. Genomics, Biotechnology, and Recombinant DNA
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The McGraw Hill Companies, 2001
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(a) Clone 1 overlapping clone 2 Restriciton sites of EcoRI 3.5 6 4 2 6.5 5 Clone 1 7 3
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(c) Regions of overlap and nonoverlap inferred from fingerprint date in (b). Fragments are arbitrarily ordered, from largest to smallest, within each region.
Figure 13.42 To create contigs, researchers must nd overlapping clones and determine
their region of overlap. In part (a), we have two overlapping pieces of DNA, found by chromosome walking. The pieces are digested with EcoRI and electrophoresed, producing the blots in part (b). From these gels, we see that fragments of 2.0, 4.0, 5.0, and 6.5 kb pairs are in common, indicating that they are in the region of overlap in both clones. We have thus isolated the overlap region and the unique end regions of both clones (compare c with a). Restriction maps can then be made of each segment, ordering the pieces. (Reprinted courtesy of Los Alamos Science, Volume Number 20, a publication of
Los Alamos National Library, Los Alamos, NM.)
Tamarin: Principles of Genetics, Seventh Edition
III. Molecular Genetics
13. Genomics, Biotechnology, and Recombinant DNA
The McGraw Hill Companies, 2001
Mapping and Sequencing the Human Genome
Figure 13.43 A contig is further built up by assembling
pairwise overlapping clones into longer sequences. Here we see that clone A overlaps clone B to the left and clone C to the right. In this case, there is one fragment common to all three clones. By comparing clones in this manner, we can march down the chromosome, creating a larger and larger contig. (Reprinted courtesy of Los Alamos Science, Volume Number 20,
a publication of Los Alamos National Library, Los Alamos, NM.)
overlap of cosmids can be determined by sequencetagged sites, RFLPs, SNPs, or microsatellites in common. The cosmids are then digested and sequenced. From the sequences we work back, nding overlap and thereby constructing a contig of that BAC. The same process is carried out on neighboring BACs, extending the contig eventually to cover the entire chromosome. At the initiation of the Human Genome Project, various goals were set. A modern linkage map of microsatellite markers of the human genome was targeted to be complete when markers were spaced about 0.7 centimorgans (about 700,000 base pairs) apart. That goal was reached in 1996 with 2,335 microsatellite markers located on the genome. The physical map of sequencetagged sites would be considered complete with markers every 100,000 bases, the equivalent of 30,000 sequencetagged sites in the genome cloned in BACs. That goal was reached in 1997. The sequence of the complete genome was targeted for 2001 and announced in 2000.
Closing the Gap Between Two Contigs
Only one walking step is needed to bridge the gap between two contigs
Contig X
Contig Y
Four walking steps are needed to bridge the gap between two contigs
Contig W
Contig Z Clone in a contig Probe used to find the next clone in a walk Next clone in a walk
Figure 13.44 When contigs of large parts of a chromosome are built up, they need to be connected. We can do this directly if there is an overlap at the end of one contig and the beginning of the next. Barring that, we must do chromosome walking to nd clones that bridge the gap between two contigs. At the top of the gure, in typical chromosome walking technique, the DNA of an end clone is fragmented and used to probe for an overlap. In this case, one clone is found that overlaps two contigs and thus joins them into one long contig. In the bottom portion of the gure, the walk requires nding four overlapping clones that bridge the gap between the two contigs. In both cases, the process is successful, joining two contigs into one longer one. (Reprinted courtesy of Los
Alamos Science, Volume Number 20, a publication of Los Alamos National Library, Los Alamos, NM.)
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
One of the reasons that goals were optimistic is that methods of mass production have been developed as the project has moved along. These methods include the automation of sequencing and cloning and the development of some new technology. For example, scientists at Affymetrix, Inc., have developed the equivalent of a DNA probe computer chip. Thousands of known DNA sequences are synthesized on a glass substrate. The DNA to be probed is introduced to this chip, where hybridization will take place. Using uorescent technology, successful probing can be determined using a laser confocal scanning system ( g. 13.45). These chips allow extremely rapid analysis of DNA sequences. Several other manufacturers have developed similar technologies. As mentioned at the beginning of this chapter, J. C. Venter of Celera Genomics was a co-announcer of the completion of the sequencing of the entire human genome.Venter and his colleagues used a whole-genome shotgun method in which the entire human genome was broken into small segments, cloned, and sequenced. The Celera group will then piece together the genome with a massive computing effort. Previously, it had been thought that this method could not work on a genome as
large as the human genome. Venter and his colleagues, however, had sequenced the Drosophila genome (180 million base pairs) by March of 2000 by this method.Venter and his colleagues had also sequenced the rst true organism, the bacterium Haemophilus in uenzae (1.8 million base pairs) in July of 1995. Since that time, the yeast Saccharomyces cerevisiae (12 million base pairs) was sequenced in 1996, and a signi cant genetic model organism, the nematode worm, Caenorhabditis elegans (97 million base pairs; see chapter 16), was sequenced in 1998. Since 1995, numerous other bacteria and eukaryotes have had their genomes sequenced.
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