Polymerase Chain Reaction

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This chapter presents many of the sophisticated techniques, developed in the past 25 years, that allow for the dissection of complex genomes into defined fragments with the complete analysis of the nucleotide sequence and function of these DNA regions. The modification and manipulation of genes, that is, genetic engineering, facilitates the introduction and expression of genes in both prokaryotic and eukaryotic cells. Many methodological approaches in genetic engineering have been greatly simplified by employment of a method that rapidly amplifies selected regions of DNA—the polymerase chain reaction (PCR). Proteins for experimental and clinical uses are readily produced by these procedures and it is anticipated that in the not too distant future these methods will allow for the rapid increase of treatment modalities of genetic diseases with gene replacement therapy. Current and potential uses of recombinant DNA technologies are also described. The significance to our society of advancements in the understanding of genetic macromolecules and their manipulation cannot be overstated.
18.2— Polymerase Chain Reaction
The rapid production of large quantities of a specific DNA sequence took a leap forward with the development of the polymerase chain reaction (PCR). The PCR requires two nucleotide oligomers that hybridize to the complementary DNA strands in a region of interest. The oligomers serve as primers for a DNA polymerase that extends each strand. Repeated cycling of the PCR yields large amounts of each DNA molecule of interest in a matter of hours as opposed to days and weeks associated with cloning techniques.
The PCR amplification of a specific DNA sequence can be accomplished with a purified DNA sample or a small region within a complex mixture of DNA. The principles of the reaction are shown in Figure 18.1. The nucleotide sequence of the DNA to be amplified must be known or it must be cloned in a vector (see p. 778) where the sequence of the flanking DNA has been established. The product of PCR is a double­stranded DNA molecule and the reaction is completed in each cycle when all of the template molecules have been copied. In order to initiate a new round of replication the sample is heated to melt the double­stranded DNA and, in the presence of excess oligonucleotide primers, cooled to permit hybridization of the single­stranded template with free oligomers. A new cycle of DNA replication will initiate in the presence of DNA polymerase and all four dNTPs. Heating to about 95°C as required for melting DNA inactivates most DNA polymerases, but a heat stable polymerase,
Figure 18.1 Polymerase chain reaction (PCR). A DNA fragment of unknown sequence is inserted into a vector of known sequence by normal recombinant methodologies. The recombinant DNA of interest does not need to be purified from contaminating DNA species. The DNA is heated to 90°C to dissociate the double strands and cooled in the presence of excess amounts of two different complementary oligomers that hybridize to the known vector DNA sequences flanking the foreign DNA insert. Only recombinant single­stranded DNA species can serve as templates for DNA replication, yielding double­stranded DNA fragments of foreign DNA bounded by the oligomer DNA sequences. The heating–replication cycle is repeated many times to rapidly produce greatly amplified amounts of the original foreign DNA. The DNA fragment of interest can be purified from the polymerase chain reaction mixture by cleaving it with the original restriction endonuclease (RE), electrophoresing the DNA mixture through an agarose gel, and eluting the band of interest from the gel.