Detection of Amplified DNAThe first detection methods used with PCR were radioactively labeled probes that identified specific amplified sequences. With improvements in specificity, it became possible to visualize amplified DNA of the predicted size directly by examining its fluorescence after staining. Probes have now been converted to nonisotopic colorimetric systems. In another approach, the probe is a 'reverse' component (bound to a membrane) and 'captures' a specific allele or a sequence variant if it is present in the amplified DNA.
Edited by: Nick A. Saunders and Martin A. Lee"an invaluable reference" (Doodys); "wide range of real time PCR technologies" (Food Sci Technol Abs); "I was impressed by this text" Aus J Med Sci read more ...
Provides both the novice and experienced user with an invaluable reference to a wide-range of real-time PCR technologies and applications and supplies detailed technical insights into the underlying principles, methods and practice of real-time PCR.
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.
- MALDI-TOF Mass Spectrometry in Microbiology
- Aspergillus and Penicillium in the Post-genomic Era
- The Bacteriocins
- Omics in Plant Disease Resistance
- Climate Change and Microbial Ecology
- Biofilms in Bioremediation
- Gas Plasma Sterilization in Microbiology
- Virus Evolution
- Aquatic Biofilms
- Thermophilic Microorganisms
- Flow Cytometry in Microbiology
- Probiotics and Prebiotics
- Corynebacterium glutamicum
- Advanced Vaccine Research Methods for the Decade of Vaccines
- Bacteria-Plant Interactions
- Metagenomics of the Microbial Nitrogen Cycle
- Pathogenic Neisseria