Basic principles of real-time quantitative PCR.
Real-time quantitative PCR allows the sensitive, specific and reproducible quantitation of nucleic acids. Since its introduction, real-time quantitative PCR has revolutionized the field of molecular diagnostics and the technique is being used in a rapidly expanding number of applications. This exciting technology has enabled the shift of molecular diagnostics toward a high-throughput, automated technology with lower turnaround times. This article reviews the basic principles of real-time PCR and describes the various chemistries available: the double-stranded DNA-intercalating agent SYBR Green 1, hydrolysis probes, dual hybridization probes, molecular beacons and scorpion probes. Quantitation methods are discussed in addition to the competing instruments available on the market. Examples of applications of this important and versatile technique are provided throughout the review.
Arya
Manit
M
Institute of Urology, Prostate Cancer Research Centre, University College London, UK. manit_arya@hotmail.com
Shergill
Iqbal S
IS
Williamson
Magali
M
Gommersall
Lyndon
L
Arya
Neehar
N
Patel
Hitendra R H
HR
D052160:Benzothiazoles; D015342:DNA Probes / Q000737:chemistry*; D003959:Diamines; D009930:Organic Chemicals / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D011804:Quinolines; D012680:Sensitivity and Specificity
D052160:Benzothiazoles; D015342:DNA Probes; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I