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Real-time PCR is based on the conventional principles of PCR and, with the simple shift of emphasis from the end-product to the whole course of the PCR process, has established itself as the most sensitive and specific quantitative PCR method. Real-time PCR is, like any other modern method in molecular genetics, expanding, with potential applications even in proteomics. With a variety of detection chemistries, an increasing number of platforms, multiple choices for analytical methods and the jargon emerging along with these developments, real-time PCR is facing the risk of becoming an intimidating method, especially for beginners. "Real-Time PCR" provides the basics, explains how they are exploited to run a real-time PCR assay, how the assays are run and where these assays are informative in real life. It doesn't cover every aspect of real-time PCR, but addresses the most practical aspects of the techniques with the emphasis on 'how to do it in the laboratory'.
1. Real-time PCR Overview. 2. Primer and Probe Design (Primer Banks). 3. Real-Time PCR using SYBR Green. 4. Normalisation. 5. Relative Quantification. 6. Analysis and Reporting 7. Allelic Discrimination Assays. 8. Methylation Assays. 9. Mitochondrial DNA Analysis. 10. Real-Time Immuno-PCR. 11. High-resolution Melting Analysis. 12. Quantification of Ocular Gene Expression. 13. Solid Organ Transplant Monitoring. 14. Real-Time PCR Applications in Haematology. 15. Clinical Microbiology (Bacterial). 16. Clinical Virology. 17. Real-time PCR for Prenatal Diagnosis of Monogenic Diseases caused by Single Nucleotide Changes - The Example of the Hemoglobinopathies.
Tevfik Dorak-Medical Department, University of Newcastle upon Tyne, Newcastle, UK