Cttaa G

synthesize DNA strands from deoxyribonucleotides, using a DNA template, as described in Chapter 25.) Isolated DNA containing the segment to be amplified is heated briefly to denature it, and then cooled in the presence of a large excess of the synthetic oligonucleo-tide primers. The four deoxynucleoside triphosphates are then added, and the primed DNA segment is replicated selectively. The cycle of heating, cooling, and replication is repeated 25 or 30 times over a few hours in an automated process, amplifying the DNA segment flanked by the primers until it can be readily analyzed or cloned. PCR uses a heat-stable DNA polymerase, such as the Taq polymerase (derived from a bacterium that lives at 90 °C), which remains active after every heating step and does not have to be replenished. Careful design of the primers used for PCR, such as including restriction endonuclease cleavage sites, can facilitate the subsequent cloning of the amplified DNA (Fig. 9-16b).

This technology is highly sensitive: PCR can detect and amplify as little as one DNA molecule in almost any type of sample. Although DNA degrades over time (p. 293), PCR has allowed successful cloning of DNA from samples more than 40,000 years old. Investigators have used the technique to clone DNA fragments from the mummified remains of humans and extinct animals such as the woolly mammoth, creating the new fields of molecular archaeology and molecular paleontology. DNA from burial sites has been amplified by PCR and used to trace ancient human migrations. Epidemiologists can use PCR-enhanced DNA samples from human remains to trace the evolution of human pathogenic viruses. Thus, in addition to its usefulness for cloning DNA, PCR is a potent tool in forensic medicine (Box 9-1). It is also being used for detection of viral infections before they cause symptoms and for prenatal diagnosis of a wide array of genetic diseases.

The PCR method is also important in advancing the goal of whole genome sequencing. For example, the mapping of expressed sequence tags to particular chromosomes often involves amplification of the EST by PCR, followed by hybridization of the amplified DNA to clones in an ordered library. Investigators found many other applications of PCR in the Human Genome Project, to which we now turn.

Genome Sequences Provide the Ultimate Genetic Libraries

The genome is the ultimate source of information about an organism, and there is no genome we are more interested in than our own. Less than 10 years after the development of practical DNA sequencing methods, serious discussions began about the prospects for sequencing the entire 3 billion base pairs of the human genome. The international Human Genome Project got underway with substantial funding in the late 1980s. The effort eventually included significant contributions from

20 sequencing centers distributed among six nations: the United States, Great Britain, Japan, France, China, and Germany. General coordination was provided by the Office of Genome Research at the National Institutes of Health, led first by James Watson and after 1992 by Francis Collins. At the outset, the task of sequencing a 3 X 109 bp genome seemed to be a titanic job, but it gradually yielded to advances in technology. The completed sequence of the human genome was published in April 2003, several years ahead of schedule.

This advance was the product of a carefully planned international effort spanning 14 years. Research teams first generated a detailed physical map of the human genome, with clones derived from each chromosome organized into a series of long contigs (Fig. 9-17). Each contig contained landmarks in the form of STSs at a distance of every 100,000 bp or less. The genome thus mapped could be divided up between the international sequencing centers, each center sequencing the mapped BAC or YAC clones corresponding to its particular segments of the genome. Because many of the

Genomic DNA

Genomic DNA

DNA is digested into fragments; fragments inserted into BACs.

Contigs are identified and mapped.

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