Polymerase
chain reaction (PCR) is a repetitive procedure that results in geometric
amplification of a specific DNA sequence. In this method, two synthetic
oligonucleotides are prepared, complementary to sequences on opposite strands
of the target DNA at positions just beyond the ends of the segment to be
amplified. The oligonucleotides serve as replication primers that can be
extended by
DNA polymerase. The 3´ ends of the hybridized probes are oriented toward each other and positioned to prime DNA synthesis across the desired DNA segment. 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 oligonucleotide 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 TaqI polymerase, 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.
DNA polymerase. The 3´ ends of the hybridized probes are oriented toward each other and positioned to prime DNA synthesis across the desired DNA segment. 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 oligonucleotide 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 TaqI polymerase, 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.
Figure: Amplification
of a DNA segment by the polymerase chain reaction.
In
addition to its usefulness for cloning DNA, PCR is a potent tool in forensic
medicine .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.
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