The polymerase chain reaction (PCR) is an in vitro method for replicating DNA. A target nucleotide sequence is copied re­peatedly so that a million copies can be made in less than an hour.

The DNA polymerase used in PCR, called Taq poly­merase, comes from a bacterium—Thermus aquaticus— that lives in hot springs; it is not denatured at high tem­peratures. Thus this DNA polymerase can withstand re­peated exposure to 95° C. This is critical because in PCR the temperature is repeatedly cycled to separate the chains of the DNA double helix, to bind the primers to the template DNA, and to allow the DNA polymerase to synthesize new strands. Each cycle lasts only a few minutes. The effect of repeated cycling is to exponen­tially increase the number of copies of a defined seg­ment of the DNA. Within an hour a single copy of a gene can be amplified to a million copies. PCR technol­ogy has applications for research and diagnosis and is fast becoming a standard procedure in biotechnology and medical diagnostic laboratories.

Polymerase Chain Reaction (pcr)

200 CHAPTER 7 MICROBIAL GENETICS: REPLICATION AND EXPRESSION OF GENETIC INFORMATION

one site, with two replication forks moving from the initiation site in opposite directions around the circu­lar bacterial chromosome. As the replication forks move around the bacterial chromosome, an en­zyme—DNA gyrase—twists the DNA. This enzyme is unique to bacteria and hence a potential site for the action of an antimicrobial agent. In fact, a new class of antibacterial agents, the quinolones, have been discovered that interfere with DNA gyrase. By pre­venting the formation of replication forks in bacterial cells, quinolones block bacterial reproduction and, hence, can be used to treat bacterial infections. The quinolone ciprofloxacin, for example, is useful in treating Pseudomonas infections.

DNA gyrase untwists the DNA of the bacterial chro­mosome.

Quinolones are antibacterial agents that inhibit DNA

gyrase.

Formation of a New Chain of Nucleotides—DNA Polymerase

Free nucleotides within the cell in association with DNA polymerase are positioned opposite their com­plementary nucleotides in the template. This process of aligning [ə'laɪnɪŋ] complementary nucleotides (A opposite T and C opposite G) is called base pairing['pɛə(r)ɪŋ]. The order of

the nucleotides is specified by the template DNA. Af­ter the nucleotides are aligned by base pairing ['pɛə(r)ɪŋ], an en­zyme called DNA polymerase links the nucleotides by forming phosphodiester bonds. The action of DNA polymerase can be likened ['laɪk(ə)n] to a zipper ['zɪpə] where the teeth of the zipper are initially aligned and pro­gressively linked together in a continuous motion.

DNA polymeras adds nucleotides to the free 3'-OH end of an existing nucleotide ['n(y)o͞oklēəˌtīd] chain of nu­cleotides (FIG. 7-10). Because DNA polymerase adds nucleotides only to the 3'-OH free end, the direction of DNA synthesis is 5'-P —3'-OH. Since the two chains of the double helical DNA molecule are an­tiparallel (one running from the 5'-P —> 3'-OH free end and the other running from the 3'-OH —» 5'-P free end) this indicates that the synthesis of the two complementary DNA chains must proceed in oppo­site directions.

One DNA chain can be continuously synthesized. It is the chain that runs in the appropriate direction for the continuous addition of new free nucleotides to the free 3'-OH end. This is the continuous or leading strand of DNA. Its synthesis occurs simulta­neously with the unwinding of the double helical molecule and progresses toward the replication fork.

Replication of dna 201

The other strand of DNA, however, cannot be syn­thesized continuously. This is because it runs 3'-OH to 5'-P but DNA polymerase only adds nucleotides in the 5'-P to 3'-OH direction. The initiation of its syn­thesis can begin only after the double helix has un­dergone some unwinding. Synthesis of this strand in­volves formation of short DNA fragments (called Okazaki fragments after the husband and wife team that discovered them) in the direction opposite the direction in which the parent DNA unwinds. Because it is synthesized discontinuously and only after syn­thesis of the continuous strand has begun, it is called

the discontinuous or lagging strand of DNA. The

short DNA fragments of the discontinuous strand are joined together by enzymes called ligases. The com­bined action of DNA polymerase and DNA ligase, thus, accomplishes the synthesis of both complemen­tary strands of DNA during replication.

To make a complementary copy of DNA, the double helix is pulled [pul] apart to form a replication fork, com­plementary nucleotides are aligned by base pairing, and phosphodiester linkages are formed by DNA polymerase.

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Sometimes a mutation results in the death of the microorganism or its inability to reproduce. This is called a lethal mutation. In other cases, the mutation alters the nutritional requirements for the progeny of a microorganism. Such a mutation is called a nutri­tional mutation. Often, nutritional mutants will be unable to synthesize essential biochemicals, such as amino acids. Auxotrophs are nutritional mutants that require growth factors that are not needed by the parent (prototroph) strain.

Replica plating is a method frequently used to de­tect auxotrophs (FIG. 7-12). In this method, bacterial cells are grown on a master plate and then trans­ferred to sterile plates by repeatedly stamping a pad over the master plate and pressing the pad into plates with media of differing composition. The distribu­tion of microbial colonies should be replicated ex­actly on each new plate. If a colony is unable to grow on the minimal media, which lacks a specific growth factor, but will grow on the complete medium, this indicates that nutritional mutants, or auxotrophs, are occurring. This method allows an investigator to screen a large number of bacteria for mutations.

FIG, 7-11 Plate showing growth of Serratia marcescens. The wild type colonies are red and the mutant colonies are

gray.

FIG. 7-12 Replica plating is used to identify mutants by transferring identical colonies to different types of media and comparing the colonies that develop on the respective plates. This method is critical in identifying auxotrophic mutants. All colonies develop on a complete medium that satisfies the nutritional needs of both the parental and mu­tant strains. Colonies of the auxotrophic mutant fail to de­velop on a minimal medium lacking the specific nutritional growth factors required by the mutant.

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