Exponential phase, of PCR

For most quantitative applications, it is important that amplification be performed during the exponential phase of PCR—that is, before the reaction... 

Real-time PCR is a quantitative method for measuring amplicons as they are produced by measuring the increase in fluorescence of a dye added to the reaction mixture.12,104,105 Methods using fluorescent reporters, such as SYBR Green,104,106 TaqMan ,107,108 or molecular beacons,9 collect quantitative data at the time when DNA is in the exponential phase of amplification. 

In addition to successful linking of target antigen and DNA marker, as discussed in the previous chapter, the subsequent amplification of the DNA is the second key factor for efficient IPCR. Similar to many protocols developed for quantitative PCR [2], the DNA amplification product has to be converted into a detectable signal. Typically, a simple yes/no decision on the presence of the DNA marker is not sufficient, and a quantitative readout dependent on the antigen concentration is needed. Therefore, in many IPCR applications the cycle number in PCR-amplification is limited to the exponential phase of the amplification for example, 30 or fewer cycles [10, 24-26, 29, 31, 33, 37]. Alternatively, successful applications of 40 cycles were also reported [34-36, 38, 39, 41], underlining the relative flexibility of PCR conditions for the amplification step. The need for an optimized cycle number is only important for end point determinations such as gel electrophoresis (Section 2.2.1) or PCR-ELISA (Section 2.2.2). Recently, the... 

PCR is primarily a qualitative technique used for detecting specific DNA or RNA sequences. Its use as a method for quantitating the relative abundance of different sequences demands careful standardisation. The dependence of c upon c0 in Eq. 5.15 is valid only for the exponential phase of the reaction, and also only when the amplification coefficients x for the different sequences are exactly the same, and remain constant over the course of the reaction. This is not usually the case even relatively minor differences in base composition can lead to differences in the amplification coefficient that are difficult to predict In addition, there is an accumulation of side products like pyrophosphate that inhibit the reaction, and also of the amplified DNA product itself which in the single stranded form competes with the primer for the complementary strand. Coupled with these factors, the concentrations of primer and dNTPs decrease as the PCR reaction progresses. The combined effect of all of these factors is that the amplification coefficient falls, the exponential relationship between cn and c0 (Eq. 5.15) collapses, and the PCR enters a plateau phase and... 

Quantitative PCR without using an internal standard has become possible with the recent devdopment of real-time thermocyclers. The advantage of this approach is that the amplified DNA is detected in the exponential phase of the amplification rather than the plateau phase, by sensitive on-line methods using fluorescent oligo-nudeotide probes or fluorescent dyes (Meuer et al. 2001). 

Immunoreagents are well suited for quantitation of amplified DNA. However, the exponential nature of PCR amplification makes it difficult to extrapolate from the amount of amplified DNA to the amount of starting material. The most reliable quantitative PCR methods involve real-time assay of amplified DNA during the exponential phase of the amplification reaction. Because the analyte of interest is usually the DNA template, not the amplification product, immunochemical methods at their present stage of development are generally best applied to an unamplified template where concentrations permit direct analysis or for qualitative assays. 

PCR is a very powerful technique, providing a sizable amount of DNA from a trace of a DNA sample. Hence it would be natural to expect that the trace amount of starting DNA can be quantitated sensitively from the amount of the finally-obtained PCR product. However, this type of quantitation based on the end-point detection is not reliable because of the saturation effects of PCR. This problem has been overcome by real-time PCR, which monitors PCR amplification in real time and enables accurate quantitation from the kinetics of the exponential phase. Real-time PCR thereby provides a highly sensitive and specific quantitation method for nucleic acids. 

The typical PCR run (Fig. 7.1) consists of three cycles denaturation (1-2 min at 3 94°C), primer annealing (1-2 min at 50-55°C), and extension (1-2 min at 72°C). This design also requires optimization for each particular PCR. The desired blunt-ended duplex product does not appear until after the third cycle, whereupon it accumulates exponentially in subsequent cycles. The number of cycles required will depend on the efficiency of the reaction per cycle. Once the desired product has reached about 1012 copies, PCR efficiency drops significantly, and product stops amassing exponentially. This is the plateau phase continuing PCR beyond this point often results in contaminating by-products rather than more product (Cha and Thilly, 1993). 

The real-time PCR fluorescence curve generated by the sequence detection system is composed of four distinct phases. When PCR product and reporter signal accumulate beyond background fluorescence levels, the reaction enters the exponential detection phase. At this point the amplification plot crosses a user-defined detection threshold which is set above the background fluorescence noise, preferable at the beginning of the exponential phase. The fractional cycle number at which the reaction crosses the threshold (C ) is related inversely to the initial template DNA concentration. As PCR products continues to accumulate, the ratio of Taq DNA polymerase to amplified products decreases, resulting in nonexponential accumulation of amplicons. At this point the reaction enters the linear phase. Once PCR product ceases to accumulate due to assay depletion, AR values remain relatively constant and the reaction enters the plateau phase. 

Real-time PCR can be used in traditional PCR applications as well as new applications that would have been less effective with traditional PCR. With the ability to collect data in the exponential growth phase, the power of PCR has been expanded into such applications as ... 

Real time RT-PCR has significantly simplified the process of producing reproducible quantitation of low-abundance mRNA and transcriptional profiling (Rajeevan et al., 2(X)1). Real time quantitation of mRNA by RT-PCR is defined by Ct (threefold cycle number) at a fixed threshold where PCR amplification is still in the exponential phase and the reaction components are not rate-limiting. Standard curves are constructed for each amplicon from which the precise copy number of mRNA transcripts is calculated or for selected ones from which the unknown sample is estimated by normalizing to the input amount of a reference gene (Aberham et al, 2001). 

PCR there is no direct relation of DNA input to amplified target hence classical RT-PCR assays have to be stopped at least in linear phase. The exponential range of amplification has to be determined for each transcript empirically by amplifying equivalent amounts of cDNA over various cycles of the PCR or by amplifying dilutions of cDNA over the same number of PCR cycles. 

In the exponential phase, the number of DNA copies increases exponentially under ideal reaction conditions. As the reaction cycles continue to increase, reagents are used and the efficiency of template amplification decreases. Amplification fails to occur in an exponential way, and the PCR enters into the plateau stage. Since it is the C, value that wiU be used for analyses, log-Unear and plateau data serve as little more than confirmation that the amplification process proceeded in a standard way. 

Although PCR amplification begins at an exponential rate, it enters a stationary phase after approximately 30 cycles, and additional cycles do not increase the concentration of amplicons. For this reason it is not practical to use PCR for quantifying bacteria directly, and other methods, such as real-time PCR, are used for this purpose. 

Figure 5-18. Dependence of yield on PCR cycle number. The amount of product increases exponentially only under optimal PCR conditions, which occur during the early cycles. Several factors cause the reaction to slow down, notably the accumulation of pyrophosphate produced in the polymerisation, increasing competition between primer and complementary DNA strand for the template, competition of the template for polymerase, and finally, dena-turation of the polymerase. The reduction in the amplification coefficient (lower) causes the initial exponential growth to enter a plateau phase where amplification ceases.

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. 

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