The Principle of PCR

The principle of PCR, including the different phases of the amplification reaction and the reagents needed, is described in the next section. This is followed by real time PCR, which allows semi-quantitative measurement of the PCR products during the reaction. Then the process of reverse transcription of RNA into cDNA is outlined. 

The amplification reaction is carried out in a single sample tube, which is placed into a thermocycler. The sample tube contains (1) an excess of two primers, i.e. oligonucleotides, that are complementary to the ends of the targeted nucleic acid region, (2) the enzyme DNA polymerase, (3) an excess of the four deoxynucleotide triphosphates (dATP, dCTP, dGTP and dTTP) (4) the template DNA, i.e. the DNA sample that is to be amplified and (5) a buffer to maintain the correct pH and to supply ions such as Mg + their are necessary for the reaction. The reaction takes place in three temperature-controlled steps (Fig. 6.2). 

In theory, the number of DNA copies is doubled during each cycle, resulting in an exponential amplification. The theoretically predicted number of DNA molecules, N, at the end of the reaction depends on the initial number of DNA copies, No, in the reaction mixture and the number of cycles, n  

However, this theoretical number is never achieved due to a number of limiting factors including the depletion of the reagents and the amplification of longer strands during the first cycles (see section 6.2.2). In practice, the number of DNA molecules can be approximated by the following equation  

For a reaction with n = 20 cycles that starts with a single DNA molecule, No = I, the number of theoretically predicted copies after the end of the reaction 

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The polymerase chain reaction (PCR) can clone (or amplify) DNA samples as small as a single molecule. If a length of DNA is mixed with the four nucleotides (A, T, C and G), and the enzyme DNA polymerase, then the DNA will be replicated many times. The principle of PCR is as follows ... 

The following discussion is focused on in vitro applications of monoclonal antibodies and nucleic acid probes as diagnostic reagents. The principles of production and characteristics of Mabs are described in Chapter 2. Their use as in vivo diagnostic imaging agents has been discussed in Chapter 3 in the context of therapeutic applications, and is not discussed further here. The principles of PCR and nucleic acid hybridization have been discussed in Chapter 2 and Chapter 4, respectively. 

Since the first publication of the PCR method (Mullis, 1990), thousands of applications for DNA analysis have been developed. The principle of PCR is shown in Figure 1.4. 

Fig. 6.2. The principle of PCR. (1) Denaturation the two DNA strands are separated at 95 °C (2) Anneahng of primers the primers are hybridised to their complementary sequences at 50-60 °C (3) Primer extension at 72 °C, the polymerase catalyses the synthesis of the complementary single stranded DNA by extending the 3 -end of the hybridised primer.

For PCR and PLS, the principle of cross validation is identical. We refer to Cross Validation (p.303). The following function PLS cross.m differs from PCR cross only in the few lines performing the calibration and calculating the prognostic vector. 

Anklam et al. [7] as well as Ahmed [8] recently published a comprehensive overview of different PCR assays that have been published in the literature. The authors tried to include performance data adding to the value of the review articles. The validation of PCR methods and thus the establishment of such performance criteria is still the subject of much debate. H bner et al. [9] suggested an approach for the validation of PCR assays. In general, it is currently the view of most researchers that validation of a PCR assay should not differ essentially from the validation of other analytical methods. Thus, all principles outlined in the ISO standard 17025 General requirements for the competence of testing and calibration laboratories, ISO standard 5725 Accuracy (trueness and precision) of measurement methods and results as well as the principles as laid down by Codex Alimentarius (http //www.co-dexalimentarius.net), are applicable to PCR. 

Tawfik and Griffith (1998) reported an in vitro selection strategy for catalytic activity using compartmentalization. Here, each member of the DNA library is encapsulated in an aqueous compartment in a water in oil emulsion. The compartments are generated from an in vitro transcription-translation system, and contain the components for protein synthesis. The dilution is chosen such that, on average, the water droplets contain less than one DNA molecule. The DNA is transcribed and translated in vitro in the presence of substrate, which is covalently attached to the DNA. Only translated proteins with catalytic activity convert the substrate to the product. Subsequently, all DNA molecules are recovered from the water droplets and the DNA linked to the product is separated from the unmodified DNA linked to the educt, which requires a method to discriminate between both. The modified DNA can then be amplified by PCR and used for a second selection cycle. The principle of this approach is depicted in Figure 6. 

In an approach similar to the cell-like compartments, Doi and Yanagawa (1999) used biotinylated DNA to display peptides fused to streptavidin in compartments of water in oil emulsions. The method was named streptavidin-biotin linkage in emulsions, STABLE (Doi and Yanagawa, 1999). Upon in vitro translation each translated peptide is displayed as a fusion to streptavidin that binds to its encoding biotinylated DNA in its compartment. The resulting protein-DNA fusions can then be recovered and used for affinity selection. To avoid cross-contamination, biotin has to be added before recovery because much more streptavidin will be produced in each compartment than biotinylated DNA is present. The selected DNA-protein complexes can then be amplified by PCR. The principle of this selection strategy is shown in Figure 7. 

The belief is that the statistical method used (such as PLS, PCR, MLR, PCA, ANNs) will extract from the data those variables which are most important, and discard irrelevant information. Statistical theory shows that this is incorrect. In particular, the principle of parsimony states that a simple model (one with fewer variables or parameters), if it is just as good at predicting a particular set of data as a more complex model, will tend to be better at predicting a new, previously unseen data set [153-155]. Our work has shown that this principle holds. 

In addition to using sequential cycles to avoid the process of serial dilution, the principle of sequential cycles can be very useful when low amounts of RNA are present. We have quantitated MDR-1 mRNA from 40 cycles in extreme cases. In that situation, SW620 is diluted to 0.12 ng input RNA since amplification 40 cycles should give a PCR product equivalent to 125 ng input RNA amplified 30 cycles. Since greater variability results from this approach, the better solution is larger biopsy samples. 

However, the real-time approach greatly reduces the interference from dATP as results are temporally dependent rather than amplitude dependent (as in more usual applications of ELIDA). Further, the use of the latest thermostable luciferases enables reagent formulations that allow nearly all NAATs (with the exception of PCR) to, in principle, be followed using the BART principle. 

Briefly outline the basic principles of PCR. Calculate the degree of amplification attained by 15 PCR cycles. 

Fig. 1. The principle of this ECL-PCR method for detection of Xanthomonas otyzae pv. Oryzicola...

The principle of in vitro selection is governed by a number of the same principles that apply to the Darwinian theory of evolution, as shown in Figure 2. First, the random sequence DNA is prepared by automated solid-phase synthesis. A mixture of four types of nucleotide is added in a stepwise condensation reaction process. When necessary, this DNA library may be converted to an RNA library by in vitro transcription or to a peptide library by in vitro translation. Second, the prepared DNA, RNA, or peptide library is subjected to affinity selection, and the molecules that bind to a target molecule are selected. Because only a very small part of the library is selected in each selection, the selected fraction is then amplified by a polymerase chain reaction (PCR) or a reverse transcription PCR (RT-PCR) technique. Successive selection and amplification cycles bring about an exponential increase in the abundance of the targeting DNA, RNA, or peptide until it dominates the population. 

Primer Extension. Primer extension uses the standard principle of PCR. Here, an oligonucleotide primer extends up to the SNP site. The assay then determines what nucleotide is incorporated into the extended PCR product by the polymerase. Often, the assay is performed in two steps. First, the genomic interval is amplified by PCR. The amplified product is subsequently mixed with another primer that anneals adjacent to the SNP site. Then individual dideoxynucleotides are added to the reaction. Upon incorporation of the matching nucleotide at the SNP site, the resulting extension product can no longer be extended further because it lacks a 3-OH group required for nucleotide incorporation. Thus, the reaction essentially stops once the nucleotide has been incorporated. 

FIGURE 2.2 Principle of PCR. As indicated PCR is mnning in cycles. Each cycle is separated in three individual steps (1, denaturation 2, annealing 3, polymerization), resulting in doubling the amount of target DNA. A typical PCR temperature-time profile is given in Table 2.2, and a standard PCR setup in Table 2.3. 

FIGURE 4.1 Principle of PCR-RFLP analysis. Amplification of conserved regions of the... 

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