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Sequencing Technologies
The ultimate physical map of the human genome is the complete DNA sequence—the determination of all base pairs on each chromosome. The completed map will provide biologists with a Rosetta stone for studying human biology and enable medical research-ers to begin to unravel the mechanisms of inherited diseases. Much effort continues to be spent locating genes; if the full sequence were known, emphasis could shift to determining gene function. The Human Genome Project is creating research tools for 21st-century biology, when the goal will be to understand the sequence and functions of the genes residing therein.
Achieving the goals of the Human Genome Project will require substantial improvements in the rate, efficiency, and reliability of standard sequencing procedures. While technologi-cal advances are leading to the automation of standard DNA purification, separation, and detection steps, efforts are also focusing on the development of entirely new sequencing methods that may eliminate some of these steps. Sequencing procedures currently involve first subcloning DNA fragments from a cosmid or bacteriophage library into special sequencing vectors that carry shorter pieces of the original cosmid fragments (Fig. 11). The next step is to make the subcloned fragments into sets of nested fragments differing in length by one nucleotide, so that the specific base at the end of each successive fragment is detectable after the fragments have been separated by gel electrophoresis. Current sequencing technologies are discussed later.
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| Fig. 11. Constructing Clones for Sequencing. Cloned DNA molecules must be made progressively smaller and the fragments subcloned into new vectors to obtain fragments small enough for use with current sequencing technology. Sequencing results are compiled to provide longer stretches of sequence across a chromosome. (Source: adapted from David A. Micklos and Greg A. Freyer, DNA Science, A First Course in Recombinant DNA Technology , Burlington, N.C.: Carolina Biological Supply Company, 1990.) |
| DNA Amplification: Cloning and
Polymerase Chain Reaction (PCR) Cloning (in vivo DNA amplification) Cloning involves the use of recombinant DNA technology to propagate DNA fragments inside a foreign host. The fragments are usually isolated from chromosomes using restriction enzymes and then united with a carrier (a vector). Follow-ing introduction into suitable host cells, the DNA fragments can then be reproduced along with the host cell DNA. Vectors are DNA molecules originating from viruses, bacteria, and yeast cells. They accommodate various sizes of foreign DNA fragments ranging from 12,000 bp for bacterial vectors (plasmids and cosmids) to 1 Mb for yeast vectors (yeast artificial chromo-somes). Bacteria are most often the hosts for these inserts, but yeast and mammalian cells are also used (a). Cloning procedures provide unlimited material for experimental study. A random (unordered) set of cloned DNA fragments is called a library. Genomic libraries are sets of overlapping frag-ments encompassing an entire genome (b). Also available are chromosome-specific libraries, which consist of fragments derived from source DNA enriched for a particular chromosome. (See Separating Chromosomes box.) |
(a)
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| (a) Cloning DNA in Plasmids. By fragmenting DNA of any origin (human, animal, or plant) and inserting it in the DNA of rapidly reproducing foreign cells, billions of copies of a single gene or DNA segment can be produced in a very short time. DNA to be cloned is inserted into a plasmid (a small, self-replicating circular molecule of DNA) that is separate from chromosomal DNA. When the recombinant plasmid is intro-duced into bacteria, the newly inserted segment will be replicated along with the rest of the plasmid. |
| (b) Constructing an Overlapping Clone Library. A collection of clones of chromosomal DNA, called a library, has no obvious order indicating the original posit-ions of the cloned pieces on the uncut chromosome. To establish that two partic-ular clones are adjacent to each other in the genome, libraries of clones containing partly overlapping regions must be constructed. These clone libraries are ordered by dividing the inserts into smaller fragments and determining which clones share common DNA sequences. | (b)
Partially cut chromosomal DNA with a frequent-cutter restriction enzyme (controlling the conditions so that not all possible sites are cut on every copy of a specific sequence) to generate a series of overlapping fragments representing every cutting site in the original sample |
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| PCR (in vitro DNA amplification) Described as being to genes what Gutenberg’s printing press was to the written word, PCR can amplify a desired DNA sequence of any origin (virus, bacteria, plant, or human) hundreds of millions of times in a matter of hours, a task that would have required several days with recombinant technology. PCR is espe-cially valuable because the reaction is highly specific, easily automated, and capable of amplifying minute amounts of sample. For these reasons, PCR has also had a major impact on clinical medicine, genetic disease diagnostics, forensic science, and evolutionary biology. PCR is a process based on a specialized polymerase enzyme, which can synthesize a complementary strand to a given DNA strand in a mixture containing the 4 DNA bases and 2 DNA fragments (primers, each about 20 bases long) flanking the target sequence. The mixture is heated to separate the strands of double-stranded DNA containing the target sequence and then cooled to allow (1) the primers to find and bind to their complementary sequences on the separated strands and (2) the polymerase to extend the primers into new complementary strands. Repeated heating and cooling cycles multiply the target DNA exponentially, since each new double strand separates to become two templates for further synthesis. In about 1 hour, 20 PCR cycles can amplify the target by a millionfold. |
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