Multiple Thermal Amplification

2.3.1. Ligase Chain Reaction In multithermal target amplification (specifically LCR), a high temperature is used to denature or unzip the double-stranded DNA (dsDNA) target

Fig. 5. linked linear amplification.

(26,27). Multiple probes complementary to different regions of the target then bind to their specific targets in the annealing step. The next step in the LCR reaction takes place at the optimal temperature for the DNA polymerase so that elongation or extension can proceed from the probes. Finally, a ligase joins the slightly extended probes together. Because LCR requires multiple cycles through all three temperatures, a thermostable enzyme capable of withstanding temperatures of 95°C and higher without becoming denatured or inactive is necessary. LCR results in the logarithmic amplification of a target sequence by doubling the target during each cycling step. The use of multiple probes in the reaction can negate potential problems arising from polymorphic regions that could adversely affect probe binding. The ligated product is then detected using a capture system. Specifically, a hapten is attached to the ends of the capture probe and the target-specific probe. A separate capture probe is removed from the reaction solution and all nonbond target probes are washed away. Only target probes bound to the capture probe can be detected using detection molecules directed to the hapten label on the 3' end of the target molecule. Figure 4B demonstrates how these amplified products are captured and detected. The closed-tube system decreases the risk for contamination and the turn around time for the assay.

2.3.2. Linked Linear Amplification Linked linear amplification (Fig. 5) is a method that is very similar to PCR except for its use of multiple nested nonreplicable primers. The non-replicable primers contain a molecule (1,3 propanediol) that prevents replication during the extension phase of the thermo-cycling reactions. Because the ends of the amplified strands cannot serve as templates for amplification, the products become progressively smaller. The main advantage of this method is its resistance to amplicon contamination because the progressively smaller products lack the primer sequences of the larger products. In addition, its ability to accommodate polymorphic targets through its use of multiple primers render it resistant to potential false-negative results resulting from polymorphic target sequences (28).

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