The PCR Amplification Process

FIGURE 3.10 Polymerase chain reaction can amplify a single molecule of DNA into millions of identical copies. 

The polymerase chain reaction has numerous applications in basic research, and its use as a diagnostic tool is expanding. The main limitation to the method is that, to synthesize appropriate oligonucleotide primers, the sequence of at least part of the molecule to be amplified must be known beforehand. 

Diagnostic applications of PCR include relatively rapid and accurate identification of pathogens of various types, including bacteria, viruses, and parasites. DNA can be amplified from samples of tissue or from biological fluids such as blood. 

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Since rolling circle amplification takes place at a constant temperature, there is no need for the target amplification process to take place in a thermal cycler, which is required to regulate the temperature for different parts of the reaction. The type of DNA polymerase to be used in RCA is not limited to thermostable enzymes, like the PCR-based diagnostics. On the other hand, the RCA method requires the environment to be free of contaminations as the RCA arrays are highly sensitive. Wiltshire [22]... 

The next major change in the analysis of the DNA for paternity (and forensic) analysis incorporated the PCR amplification of microsatellites instead of minisatellites. Microsatellites are also formed by tandem repeats but consist of two to five nucleotides per repeat units. This means that the amplification requires less DNA (less than 1 ng) and the quality of the material may be less than ideal. This capability permits the analysis of some degraded DNAs. The potential number of loci is very large and the process is rapid it may be completed in a day or two. This system also has the benefit of lending itself to multiplexing and automation. In addition, several kits are available, and for some multiplexes inexpensive silver stain materials may be employed without expensive equipment. 

FIGURE 3.2 Principle of the widely used TaqMan technology. Shown is only one cycle of the entire PCR amplification process. After denaturation and anneaUng of primers and probes, the Taq DNA polymerase synthesizes a new DNA strand until the probe is reached. Due to its 5 nuclease activity, the oUgonucleotide is cleaved and the probe is released. 

The most widely used DNA polymerase in PCR is from Thermus acquaticus, the Taq polymerase. This enzyme has an optimal temperature for polymerization in the range of 70 to 75°C. It extends DNA chains at a rate of about 2 kb per minute. It is fairly resistant to the continual cycles of heating and cooling required for PCR. The half-life for thermal denaturation of Taq polymerase is 1.6h at 95°C. When very high denaturation temperatures are needed, as in the PCR amplification of very G+C-rich DNA, more thermal-stable polymerase such as the enzyme from Thermococcus litoralis with a half-life of 1.8h at 100°C or the enzyme from Pyrococcus furiosis with a half-life of 8 h at 100°C, can be employed. However, these enzymes are not as processive as Taq polymerase, which makes it more difficult to amplify long templates. 

The introduction of fluorescent chemistries into reaction systems has made it possible to detect PCR product concentration through its relation to fluorescence intensity. The fluorescent chemistries that are used in real-time quantitative PCR will be introduced in detail later. First, a discussion of the DNA amplification process is needed to acquire a basic understanding of the principles underlying PCR. 

These reference genes demonstrate that the DNA isolated was of sufficient quality and quantity for PCR amplification. It is assumed that in the course of food processing, the species-specific reference gene and the transgene are degraded in a similar manner. It is also assumed that effects of the matrix on PCR amplification will be similar. The reduced amplification efficiency of both genes presumably has no effect on the ratio of their amounts, which reflects the ratio of modified and unmodified DNA. 

The total amplification achieved by PCR is described by the expression, (1 + )", where E is the average per-cycle efficiency and n is the total number of cycles. The amount of target sequence and the variable presence of inhibitors in clinical specimens influence both the efficiency and the kinetics of amplification. As seen in the preceding expression, small differences in the efficiency of amplification are exponentially compounded and lead to very large and unpredictable differences in product yield. The situation is even more complicated when the target is RNA. PCR must be preceded by reverse transcription to produce complementary DNA (cDNA), and the efficiency of this process is another variable that may influence product yield. 

The PCR phase of such a study is very similar to PCR amplification of DNA in a reaction tube placed in a thermocycler. Here, however, the specimen is affixed to a slide covered with the reactants (buffer, DNTPs, primers, and a thermostabile DNA polymerase) and placed on the heating block of a traditional or modified (for slides) thermocycler programmed to provide the optimum temperatures for denaturation, primer annealing, and extension. After amplification for 20-30 cycles, the specimen is processed for ISH. 

Fig. 32.1. PCR reactor for the real-time electrochemical detection of Salmonella enterica serovar Typhimurium ATCC 14028 based on the doubly labeled PCR amplification performed with the magnetic bead primer. White dots show the non-specific electrochemical signal processing the negative PCR control, while the black dots show the increasing signal of DNA IS200 doubly labeled amplicon onto magnetic beads. In all cases, 60 pg AntiDig-HRP were used. Other experimental details are medium, phosphate buffer 0.1 mol L-1, KC1 0.1 mol L-1, pH 7.0 mediator, hydroquinone 1.81 mmol L 1 substrate, H202 4.90 mmol L 1 applied potential = -0.1 V (vs. Ag/AgCl).

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