The Polymerase Chain Reaction

Once the gene has been isolated, the next step is to join it to a molecular on-switch, a sequence of DNA that will allow the cell to use the gene to make the desired protein. The on-switch, called a promoter, is matched to the type of cell that will be used for production. 

Polymerase chain reaction (PCR). Oligonucleotides complementary to a given DNA sequence prime the synthesis of only that sequence. Heat-stable Taq DNA polymerase survives many cycles of heating. Theoretically, the amount of the specific primed sequence is doubled in each cycle. Sign in atwww.thomsonedu.com/ iogin to see an animated version of this figure. 

Amplification of the amounts of DNA in extremely small samples has made it possible to obtain accurate analyses that were not possible earlier. Forensic applications of the technique have resulted in positive identifications of crime victims and suspects. Even minuscule amounts of ancient DNA, such as those available from Egyptian mummies, can now be researched after amplification. The following Biochemical Gonnections box describes some forensic uses of DNA technology. 

Polymerase chain reaction (PGR) is a sophisticated, automated technique for amplifying DNA from very small amounts of sample. 

The DNA to be amplified is mixed with specific primers, dATPs, dGTP, dGTP, dTTP, and a heat-stable form of DNA polymerase. 

The mixture undergoes 20 to 40 rounds of DNA polymerization via cycling the temperatures so that the DNA strands separate, the primers anneal, and the polymerase fills in the DNA. Each cycle doubles the target DNA. 

Mullis, K. B., The polymerase chain-reaction (Nobel Lecture) , Angew. Chem., Int. Ed. Engl. 1994, 33, 1209-1213. 

PCR works by the synthesis of two short oligonucleotides that bind correctly to opposite strands of the DNA to be replicated. This allows the DNA polymerase enzyme to begin to assemble copies of the two strands, resulting in two new DNA strands. Heating the sample causes the unwinding of the resulting double helices and provides four fresh strands that can be used as the templates for the formation of four more strands. The procedure is repeated 20-60 times over the course of a few hours either 

Synthetic oligonucleotide with altered genetic code word (GGG instead of GAG) 

One area of basic biochemical research that has paid unexpected dividends is DNA replication. Enzymological work here has characterized the various DNA polymerases in bacterial and eukaryotic cells. With progress in the biochemical characterization of these enzymes, new applications have been found for them in research 

Question How is it possible to obtain sufficient quantities of a particular DNA sample to enable a determination of its properties  

This can be achieved using the polymerase chain reaction (PCR) which is indeed a type of chain 

The polymerase chain reaction uses (1) a thermostable DNA polymerase, such as Taq polymerase derived from the bacterial thermophile Thermus aquaticus, (2) a DNA template which is to be amplified, (3) two primers, each typically of around 20 nucleotides, which anneal to distinct parts on the complementary strands of the target and serve as sites for commencing DNA polymerase action, (4) a solution including the four deoxynucleoside triphosphates dATP, dCTP, dGTP and dTTP, Mg2+, salts and pH buffer. 

PCR uses DNA polymerase to make complementary copies of DNA corresponding to the region of interest. To direct the enzyme to the correct sequence, PCR relies on primers which anneal to complementary sequences in target single-stranded DNA. DNA polymerase cannot start DNA synthesis de novo, and can extend the chain only from the annealed primers. An excess of these primers is provided in the reaction mixture to allow sufficient material for generating amplified product. 

PCR reactions include the cyclical use of high temperatures which can lead to the denaturation of thermolabile enzymes for this reason a thermostable DNA polymerase, Taq polymerase, is typically used. 

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The plaque assay is desirable because it is very sensitive and only detects infectious viral particles. However, there are viral agents which cannot be supported by cell lines. In these cases other methods must be used. The polymerase chain reaction (PGR), which amplifies DNA or RNA from viral agents, can be used to detect the presence and quantity of viral agents. The amount of RNA or DNA target in the initial sample can be determined by competitive PGR where the quantity of amplified product is compared to a control PGR product where the initial amount of target is known. Quantification is also possible by an end-point dilution method similar to that used to determine a tissue culture infections dose. PGR methods can be very sensitive however. 

FIGURE 28.14 The polymerase chain reaction (PCR). Three cycles are shown the target region appears after the third cycle. Additional cycles lead to amplification of the target region. 

K. B. Mullis (La Jolla, California) invention of the polymerase chain reaction. 

Figure 28.9 The polymerase chain reaction. Details are explained in the text.

I been established to serve as a registry of convicted offenders. When a DNA sample is obtained from a crime scene, the sample is subjected to cleavage with restriction endonucleases to cut out fragments containing the STR loci, the fragments are amplified using the polymerase chain reaction, and the sequences of the fragments are determined. 

Sequencing of DNA is carried out by the Sanger dideoxy method, and small DNA segments can be synthesized in the laboratory by automated instruments. Small amounts of DNA can be amplified by factors of 106 using the polymerase chain reaction (PCR). 

While many diseases have long been known to result from alterations in an individual s DNA, tools for the detection of genetic mutations have only recently become widely available. These techniques rely upon the catalytic efficiency and specificity of enzyme catalysts. For example, the polymerase chain reaction (PCR) relies upon the ability of enzymes to serve as catalytic amplifiers to analyze the DNA present in biologic and forensic samples. In the PCR technique, a thermostable DNA polymerase, directed by appropriate oligonucleotide primers, produces thousands of copies of a sample of DNA that was present initially at levels too low for direct detection. 

Figure 40-7. The polymerase chain reaction is used to amplify specific gene sequences. Double-stranded DNA is heated to separate it into individual strands. These bind two distinct primers that are directed at specific sequences on opposite strands and that define the segment to be amplified. DNA polymerase extends the primers in each direction and synthesizes two strands complementary to the original two. This cycle is repeated several times, giving an amplified product of defined length and sequence. Note that the two primers are present in excess.

There have been a number of attempts to achieve this objective, but so far the challenge has not been fully met. This Chapter will examine some of the conventional approaches and then go on to consider how recent developments in the use of the Polymerase Chain Reaction (PCR) with DNA for the identification of species and individual organisms by DNA analysis, sometimes known as DNA Fingerprinting", have identified a yet unrealized need for a new dimension of certified reference materials. 

Kasai K, Nakamura Y and White R 1990) Amplification of a variable number of tandem repeats (VNTR) locus (pMCTii8) by the polymerase chain reaction (PCR) and its application to forensic science. ] For Sci 35 1196-1200. 

Wang AM, Doyle MV, and Mark DF (1989) Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sd USA 86 9717-9721. 

Synthetic oligonucleotides are very important tools in the study and manipulation of DNA, including such techniques as site-directed mutagenesis and DNA amplification by the polymerase chain reaction. The techniques for chemical synthesis of oligonucleotides are highly developed. Very efficient automated methodologies based on solid phase synthesis are used extensively in fields that depend on the availability of defined DNA sequences.52... 

Progress in molecular biology has provided a new perspective. Techniques such as the polymerase chain reaction and single-strand conformation polymorphism analysis have greatly facilitated the molecular analysis of erythroenzymopathies. These studies have clarified the correlation between the functional and structural abnormalities of the variant enzymes. In general, the mutations that induce an alteration of substrate binding site and/or enzyme instability might result in markedly altered enzyme properties and severe clinical symptoms. 

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