Rolling Circle Amplification Technology RCAT

Several novel methods other than PCR have been proposed for amplifying DNA. These methods all use DNA polymerase to amplify DNA but they avoid the high temperature requirement for DNA denaturation and the consequent temperature cycling. Which of these methods will prove useful in the long run is still undecided. Perhaps quencher Molecule that prevents fluorescence by binding to the fluorophore and absorbing its activation energy

On Site Diagnosis of Plant Disease by Realtime PCR

The development of realtime PCR has resulted in a great decrease in the time needed for detection of DNA from infectious microorganisms. Classical methods often required 3 or 4 days to isolate the microorganism and another week to confirm its identity—always assuming the pathogen can be cultured. Standard PCR methods not only cut the time needed to 2 to 3 days but also work directly on tissue samples without requiring that the microorganisms should be cultured. Realtime PCR with fluorescent detection has cut the time required for diagnosis even further, although the technique was originally lab-based and relatively expensive. However, portable realtime PCR machines have been developed recently that allow DNA identification in a couple of hours. An example is the Smart Cycler® TD made by Cepheid Corporation of Sunnyvale, California.

The Smart Cycler® has been used to diagnose plant diseases, on-site in the fields where the crops are growing. For example, watermelon fruit blotch is a bacterial disease that causes major economic losses of watermelon crops worldwide. The causative agent, Acidovorax avenae subsp. citrulli requires 10-14 days for diagnosis by classical procedures. Portable, realtime PCR allows on-site identification within an hour of taking samples from plants with suspected infections. This rapid diagnosis is of great value both in managing crop diseases and also in deciding whether quarantine is required when facing a possible outbreak of a transmissible plant disease that could threaten crops in other locations.

FIGURE 23.24 Portable Realtime PCR

The Cepheid Smart Cycler® system has been used for rapid on-site detection of plant diseases. Courtesy Cepheid Corporation.

FIGURE 23.24 Portable Realtime PCR

The Cepheid Smart Cycler® system has been used for rapid on-site detection of plant diseases. Courtesy Cepheid Corporation.

FIGURE 23.25 Scorpion Primer with Combined Fluorescent Probe

Scorpion primers provide another method to detect the PCR product by fluorescence. The Scorpion probe has a stem loop structure that keeps the fluorophore molecule (diamond) in close proximity to the quencher (circle). The loop has a sequence complementary to the target DNA. The stem/loop is linked to a re gular PCR primer designed to amplify the target DNA. During the extension step of PCR, the primer portion of the probe anneals to the template and Taq polymerase makes new DNA. During the next denaturation step, the whole probe plus new DNA strand become single-stranded. The loop (blue) can now anneal to the single-stranded target DNA, releasing the fluorophore from the quencher. The fluorescence emitted is a direct measure of the amount of PCR product produced.

Scorpion probe

Scorpion probe

Fluorophore Quencher

Target DNA

HEAT TO DENATURE, THEN COOL AND ANNEAL PRIMERS

Extension phase

2~ttfm

New DNA

HEAT to denature

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