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In the past, in many instances (in museum specimens, dried specimens, crime scene evidence, and fossils), a DNA sample was available, but in such small quantity or so old as to be considered useless for study. That situation changed in 1983 when Kary Mullis, a biochemist working for the Cetus Corporation, devised the technique we now refer to as the polymerase chain reaction (PCR). PCR can be used to amplify whatever DNA is present, however small in quantity or poor in quality. The only requirement is that the sequence of nucleotides on either side of the sequence of interest be known. That information is needed to construct primers on either side of the sequence of interest. Once that is done, the sequence between the primers can be ampli ed.
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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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Genomics, Biotechnology, and Recombinant DNA
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In the PCR technique, the primers and the ingredients for DNA replication are added to the sample. Then, the mixture is heated (e.g., 95 C for twenty seconds) to denature the DNA. The temperature is then lowered (e.g., 55 C for twenty seconds) so that primers can anneal to their complementary sequences. The temperature is then raised again (e.g., 72 C for twenty seconds) for DNA replication. Then, a new cycle of replication is initiated ( g. 13.30). The various stages in the cycle are controlled by changes in temperature since the temperatures for denaturation, primer annealing, and DNA replication are different. About twenty cycles of PCR produces a million copies of DNA; thirty cycles make a billion copies. The technique is aided by using DNA polymerase from a hot-springs bacterium, Thermus aquaticus, that can withstand the denaturing temperatures. Thus, after each cycle of replication, no new components have to be added to the reaction mixture. Rather, the cycling can be continued without interruption in PCR machines (simply programmable water baths that accurately and rapidly change the water temperature that surrounds the reaction mixture). Some machines can process ninety-six samples at a time. PCR has been used to create DNA ngerprints by amplifying microsatellite DNA. These are repeats of very short sequences of DNA dispersed throughout the genome. For example, cytosine-adenine (CA) repeats occur tens of thousands of times in eukaryotes, in repeats of from twenty to sixty base pairs. As in the case of VNTR loci, there is tremendous variability among people in the number of these repeats at a locus, due presumably to crossover errors. Unlike the situation with VNTR loci, however, PCR ampli cation of one of these loci can be done without restriction cutting, Southern blotting, and probing PCR gives the results directly upon electrophoresis. All we need are the surrounding primer sequences to any microsatellite locus. PCR is now a routinely used tool in the laboratories of molecular geneticists. They use it to rapidly amplify the DNA regions of interest for research or forensic uses.
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5 Cycle 1 Steps 1 and 2
Step 3
Cycle 2 Steps 1 and 2
Step 3
Cycle 3 Steps 1 and 2
Figure 13.30 Polymerase chain reaction. DNA is denatured,
(step 1), primer oligonucleotides that are complementary to end sequences on the two strands anneal (step 2), and DNA replication takes place (step 3). Each step in the cycle is controlled by temperature changes. The targeted sequence is shown as red on one stand and blue on the other. Primers are shown as either green or yellow lollipops. A green primer begins the copying of the red strand into a complementary blue strand; a yellow primer begins the copying of a blue strand into a complementary red stand. In three cycles, one double-stranded region of DNA becomes eight. The process requires the addition of primers, deoxynucleotide triphosphates, and DNA polymerase, as well as changing temperature cycles.
Step 3
Tamarin: Principles of Genetics, Seventh Edition
III. Molecular Genetics
13. Genomics, Biotechnology, and Recombinant DNA
The McGraw Hill Companies, 2001
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