Sequence detection system

Streptavidin-single-stranded DNA covalent conjugates were described as the building blocks for assembling nanostructured scaffolds [31], The amount and type of biotinylated ligands were used to modulate the affinity of duplex formation between solid-phase-bound nucleic acid templates and DNA-streptavidin conjugates. This system has been proposed for the design of fine-tuned sequence detection systems. 

Figure 6. Representative real-time PCR results generated using an ABI 5700 sequence detection system and sequence-specific primers for a putative 0-methyl transferase. Upper panel SYBR Green I signals generated fiom root hair cDNA vs. non-root hair cDNA. Lower panel same cDNAs used as in upper panel showing signals generated fix>m an internal control gene (18S ribo-somal RNA).

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. 

Detection is carried out using a real-time PCR system (7900 HT Sequence Detection System, Applied Biosystems or equivalent) that unlike gel electophoresis, has the advantage that it is possible to see the results during amplification. The procedure is as follows ... 

ABI Prism 7700 or 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA) and associated data analysis software. 

Applied Biosystems Prism 7000 Sequence Detection System (Applied Biosystems). 

Mix/DNA sample to the reaction plates. Perform real-time PCR detection of dotA using an ABI Prism 7000 Sequence Detection System (Applied Biosystems) or comparable instrument. Extrapolate the number of C. burnetii genomes present in the DNA sample from the standard curve. 

Livak KJ (1997 and 2001) ABI Prism 7700 Sequence detection System User Bulletin 2 Relative quantification of gene expression, http //docs.appliedbiosystems.com/pe-biodocs/04303859.pdf. 

PCR conditions. The Perkin-Elmer ABI Prism 7700 sequence Detection System was used for real-time analyses. Thermal cycling conditions were specified as follows initial melting at 98°C for 10 minutes, followed by 40 cycles of melting at 95°C for 1 minute, annealing at 57°C for 1 minute and extension at ITC for 1 minute. These reactions ended with a final extension step at 72 C x 5 minutes. Fluorescent measurements were recorded during each annealing step. At the end of each PCR run, data were automatically analyzed by the system and amplification plots were obtained. 

Propagating events or propagating factors allow the accident sequence to continue, developing consequences that are subsequently more severe. Examples are failures of release-detection systems or the presence of ignition sources. 

Fig. 7.1S Schematic layout of mercury detection system showing valve sequencing for collection, flush and measurement.

In this example, there are two detection systems and two reaction opportunities. These yield three paths that lead to no adverse consequences and four paths that lead to failure with overflow as the consequence. The point is that sometimes there are more opportunities for things to go wrong than to go right. When a system or process fails, it may he difficult to trace the reasons for its failure. Based on available historic incident data, the anatomy of a major incident is rarely simple and rarely results from a single root cause. Serious incidents typically involve a complex sequence of occurrences and conditions. This sequence can include ... 

FIGURE 3.1 Development of PCR-based detection systems for mycotoxin producing-fungi from 1996 until April 2007. First publication of a diagnostic primer pair was taken as the time mark for each species depicted. In many cases, more systems were published later for the same fungus. Mycotoxin biosynthesis genes used as sequence source are given in bold. 

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