Real Time Pcr
Contents REAL TIME PCR 2 REAL-TIME QUANTITATIVE PCR (qPCR) 2 BASIC PRINCIPLE 3 TYPES OF PCR 4 qPCR STEPS 4 ONE-STEP OR TWO-STEP REACTION 6 Overview of qPCR and qRT-PCR components 6 REAL TIME PCR SYSTEM: 7 SOFTWARES FOR DATA ANALYSIS AND PRIMER DESIGNING 8 STANDARD REAL-TIME PCR PROTOCOL 9 ASSAY DESIGN 9 2. Nucleic acid purification 9 3. Reverse transcription 9 4. Controls and normalization 9 5. Standard curve evaluation of efficiency, sensitivity, and reproducibility 9 Real-Time PCR Fluorescence Detection Systems 12 DNA-Binding Dyes 12 Primer-Based Detection Systems 13 PROBE-BASED DETECTION SYSTEMS 14 Hybridization probes (also called FRET probes) 16 MELTING CURVE ANALYSIS 16 Multiplex real-time PCR 18 APPLICATIONS OF REAL TIME PCR 18 GENE EXPRESSION ANALYSIS 18 SNP GENOTYPING 19 HIV DETECTION 19 CYSTIC FIBROSIS (CF) DETECTION: 20 THE ADVANTAGES OF REAL-TIME PCR 20 THE DISADVANTAGES 21 REFRENCES 21
REAL TIME PCR
TRADITIONAL PCR
The polymerase chain reaction (PCR) is one of the most powerful technologies in molecular biology. Using PCR, specific sequences within a DNA or cDNA template can be copied, or “amplified”, many thousand- to a millionfold. In traditional (endpoint) PCR, detection and quantitation of the amplified sequence are performed at the end of the reaction after the last PCR cycle, and involve post-PCR analysis such as gel electrophoresis and image analysis.
REAL-TIME QUANTITATIVE PCR (qPCR)
In real-time quantitative PCR (qPCR), the amount of PCR product is measured at each cycle. This ability to monitor the reaction during its exponential phase enables users to determine the initial amount of target with great precision.
WHAT’S WRONG WITH AGAROSE GELS? * Poor precision. * Low sensitivity. * Short dynamic range < 2 logs. * Low resolution. * Non-automated. * Size-based discrimination only * Ethidium bromide staining is not very quantitative
REAL TIME PCR
TRADITIONAL PCR
The polymerase chain reaction (PCR) is one of the most powerful technologies in molecular biology. Using PCR, specific sequences within a DNA or cDNA template can be copied, or “amplified”, many thousand- to a millionfold. In traditional (endpoint) PCR, detection and quantitation of the amplified sequence are performed at the end of the reaction after the last PCR cycle, and involve post-PCR analysis such as gel electrophoresis and image analysis.
REAL-TIME QUANTITATIVE PCR (qPCR)
In real-time quantitative PCR (qPCR), the amount of PCR product is measured at each cycle. This ability to monitor the reaction during its exponential phase enables users to determine the initial amount of target with great precision.
WHAT’S WRONG WITH AGAROSE GELS? * Poor precision. * Low sensitivity. * Short dynamic range < 2 logs. * Low resolution. * Non-automated. * Size-based discrimination only * Ethidium bromide staining is not very quantitative