Amplicon, increase

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. 

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. 

Fig. 31.3. Rapid electrochemical verification of PCR amphfication of Salmonella enterica serovar Typhimurium ATCC 14028 with the doubly labeled primer set IS200, showing an increasing amount of IS200 doubly labeled amplicon (from 2.8 to 75.4 fmol). The negative PCR control is also shown. 60 pg AntiDig-HRP and 6.2 x 106 magnetic beads 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 = —0.1V (vs. Ag/AgCl). All data are given as average +SD, n — 3.

Multiplex PCR of the muscular dystrophin gene was performed on-chip. The method of degenerate oligonucleotide-primed PCR (DOP-PCR) was first used to increase the number of DNA templates before multiplex PCR. Therefore, DOP-PCR can provide the template DNA from the whole human genome, but this procedure is feasible only when the amplicon size is less than 250 bp [931]. 

Mixed Mechanism Probes. Several probe systems appear to function by both hydrolysis and hybridization mechanisms. These include hairpin probes, self-probing amplicon primers, and displacement probes. A hairpin probe functions similarly to a hairpin primer in that it is designed to increase in fluorescence when the distance between the quencher and the reporter increases upon target hybridization (see Figure 37-24, row five). Similarly, primers that... 

OLA. The OLA uses an enzymatic reaction to increase the specificity of a hybridization-based approach. Three very specific oligonucleotide probes are used in OLA one specific for the wild-type allele, one specific for the variant allele, and a common probe that carries a fluorescent label. PCR is used to create amplicons containing the polymorphic site. When the PCR products are incubated with all three probes, the 5 region of the common probe anneals just downstream of the polymorphic site. The 3 end of either of the allele specific probes anneals adjacent to the 5 end of the common probe. In the presence of thermostable DNA ligase, the two probes will join only if there is a perfect match. The results of the assay can be observed either by gel... 

Analysis of the effect of varying the amount of target template demonstrated a key property of BART. As the starting copy number of template decreases, the time taken to reach the maximum of light intensity increases (Fig. 3). Using agarose gel electrophoresis we were able to confirm that the time to light peak is proportional to the amount of DNA amplicon produced. As such, BART is demonstrated to be fully quantitative. 

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