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: David Rodríguez-Lázaro"I would recommend this text to anyone" (AJMS); "an excellent, detailed guide" (Emerg. Inf. Dis.) read more ...
An indispensable manual on real-time PCR for scientists in the food industry and for anyone involved in the detection of foodborne pathogens.
Edited by: Martin Filion"useful book ... filled with valuable information" (Doodys); "an outstanding book" (Fungal Diversity) read more ...
Aimed specifically at microbiologists, this volume describes and explains the most important aspects of current real-time quantitative PCR (qPCR) strategies, instrumentation and software.
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.
- Advanced Vaccine Research Methods for the Decade of Vaccines
- Bacterial-Plant Interactions
- Metagenomics of the Microbial Nitrogen Cycle
- Pathogenic Neisseria
- Human Pathogenic Fungi
- Applied RNAi
- Molecular Diagnostics
- Phage Therapy
- Bioinformatics and Data Analysis in Microbiology
- The Cell Biology of Cyanobacteria
- Pathogenic Escherichia coli
- Campylobacter Ecology and Evolution
- Next-generation Sequencing
- Omics in Soil Science
- Applications of Molecular Microbiological Methods
- Genome Analysis
- Bacterial Toxins