Detection of Amplified DNA
An alternative to probe-based detection system relies on labeled primers and strives for perfect target specificity in the amplification reaction. This process is straightforward if the target gene differs from the unintended targets by a deletion or gene rearrangement. Using the Duchenne muscular dystrophy gene deletions as a model, researchers have now automated this method. It should prove highly useful in forensic investigations for rapid analysis of amplified targets that differ in length, such as variable number tandem repeat (VNTR) loci.
In general, nonradioactive detection systems fall into two classes, direct and indirect, based on the detectability of the label. In most indirect detection methods, the primary label (e.g., biotin) is identified through its interaction with a secondary system that contains a detectable reporter group. Various techniques for direct detection of nucleic acids include the following:
1. Direct enzymatic detection, which requires the construction of enzyme DNA conjugates.
2. Fluorescent detection, which depends on the ability to synthesize fluorescent DNA. This technique may emerge as the detection technology of choice in future PCR systems.
3. Chemiluminescent detection via direct attachment of chemiluminescent labels (e.g., acridium esters and isoluminol derivatives) to synthetic nucleotides.
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