Polymerase Chain Reaction
A method has been devised with such remarkable sensitivity that it can detect a single molecule of a specific sequence. Furthermore, the single molecule can be detected in the presence of a 106 or greater excess of other sequences. This method is called the polymerase chain reaction or PCR. The polymerase chain reaction is also useful for studying specific genes or sequences. For example, it permits the sequencing in a day or two of a stretch of several hundred nucleotides with the starting point being a small sample of blood. No cloning is required for the sequencing. Such sensitivity permits the rapid characterization of the basis of mutations or of genetic defects. This extraordinary sensitivity also provides a sensitive test for the presence of a virus like HIV. Again a sample of blood can be taken and the assay can detect the presence of one copy of the single virus sequence in 100,000 cells. The polymerase chain reaction greatly facilitates generation of mutants in vitro and the synthesis of DNA for physical experiments.
Figure 10.7 Polymerase chain reaction to amplify the sequence containedbetween the sites to which two primers hybridize.
The polymerase chain reaction is a scheme that amplifies the DNA lying between two sequences which are within several thousand base pairs of one another. The amplification is accomplished by first dena-turing the DNA sample, hybridizing two oligonucleotide primers to the DNA, elongating with DNA polymerase, and repeating this cycle up to 40 times (Fig. 10.7). The two oligonucleotide primers must be comple-mentary to opposite strands of the DNA. The product of elongation primed by one oligonucleotide plus the template can then become templates for the next round of synthesis. As a result, each round of synthesis doubles the number of product DNA molecules present. The first round of synthesis produces DNA extending in one direction beyond each primer, but the DNA made in subsequent cycles from the first product DNA extends just to the ends of the primers.
Although DNA polymerase I from E. coli could be used in the polymerase chain reaction, its use would be inefficient because each round of denaturing the double-stranded DNA to form the single strands necessary as templates would destroy the polymerase. Therefore, these procedures use a temperature-resistant polymerase isolated from a thermophile, Thermus aquaticus. This polymerase withstands the 95° incubation for denaturing the DNA. Even better, after an incubation at 45° to hybridize primer to the DNA, a temperature at which the polym-erase is largely inactive, the temperature can be raised to 75° to activate the polymerase. Although a little of the primer dissociates from the template at this temperature, a much greater fraction of any incorrectly hybridized primer dissociates from incorrect sites. Thus, a very great specificity is achieved for amplification of just the desired sequence of DNA.
The polymerase chain reaction can be put to a wide variety of uses. One simple example is the screening of cloning steps. Ordinarily, one must screen transformants after the simple step of inserting a DNA fragment into a plasmid. Typically 90% of the transformants contain the fragment, but since the frequency is not 100%, one must verify that
Figure 10.8 Polymerase chain reaction used to monitor the insertion of aforeign DNA between the sites to which two primers hybridize. Insertion of DNA between the primer hybridization sites increases the size of the PCR product.
the clone selected for further work is suitable. Previously such verifica-tion required growth of cultures from a collection of candidates, puri-fication of the plasmids from each, and screening of each by restriction enzyme digestion followed by gel electrophoresis to check for the restriction fragment with properly altered size. PCR permits the same test in much less time. Transformant colonies are picked directly into tubes used for PCR. The first heat step lyses the cells. Primers are used that flank the site into which the fragment was to be cloned. The product of the amplification is run on a gel. If the fragment had been cloned between the sites, the amplified piece will have one size, but if the fragment had not been cloned, the fragment would be much smaller (Fig. 10.8).
DNA for footprinting or sequencing can be made directly from genomic DNA with the polymerase chain reaction. By labeling one of the oligonucleotide primers used in the PCR reaction, the DNA that is synthesized is already radioactive and ready for use. Such a technique eliminates the need for cloning the DNA from mutants. It also stream-lines the screening of genetic diseases or of mutants isolated in the lab. Instead of cloning the DNA to determine the defect in a gene, the DNA from the organism or cells can be directly amplified and the defect determined by sequencing. PCR also greatly facilitates genetic construc-tions. For example, suppose a portion of a gene is to be cloned in an expression vector. The oligonucleotide primers can include not only regions homologous to the DNA to be cloned, but also additional regions necessary for cloning and expression like restriction sites, ribosome binding sites, and translation termination signals (Fig. 10.9). In the first round of amplification, the regions of homology between the primers and the template DNA hybridize. In subsequent rounds, the entire primer base pairs and amplified DNA constitutes the fusion of the sequence plus the desired flanking sequences.
Figure 10.9 Use of PCR to both amplify a selected region of DNA as well asplace specific desired sequences on the ends of the DNA.
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