REAL TIME PCR2
REAL-TIME QUANTITATIVE PCR (qPCR)2
TYPES OF PCR4
ONE-STEP OR TWO-STEP REACTION6
Overview of qPCR and qRT-PCR components6
REAL TIME PCR SYSTEM:7
SOFTWARES FOR DATA ANALYSIS AND PRIMER DESIGNING8
STANDARD REAL-TIME PCR PROTOCOL9
2. Nucleic acid purification9
3. Reverse transcription9
4. Controls and normalization9
5. Standard curve evaluation of efficiency, sensitivity, and reproducibility9
Real-Time PCR Fluorescence Detection Systems12
Primer-Based Detection Systems13
PROBE-BASED DETECTION SYSTEMS14
Hybridization probes (also called FRET probes)16
MELTING CURVE ANALYSIS16
Multiplex real-time PCR18
APPLICATIONS OF REAL TIME PCR18
GENE EXPRESSION ANALYSIS18
CYSTIC FIBROSIS (CF) DETECTION:20
THE ADVANTAGES OF REAL-TIME PCR20
REAL TIME 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.
* Size-based discrimination only
* Ethidium bromide staining is not very quantitative
REAL TIME PCR VS PCR
Quantitative PCR is carried out in a thermal cycler with the capacity to illuminate each sample with a beam of light of a specified wavelength and detect the fluorescence emitted by the excited fluorochrome. The thermal cycler is also able to rapidly heat and chill samples thereby taking advantage of the physicochemical properties of the nucleic acids and DNA polymerase.The PCR process generally consists of a series of temperature changes that are repeated 25 – 40 times, these cycles normally consist of three stages: the first, at around 95 °C, allows the separation of the nucleic acid’s double chain; the second, at a temperature of around 50-60 °C, allows the alignment of the primers with the DNA template; the third at between 68 - 72 °C, facilitates the polymerization carried out by the DNA polymerase
In real-time PCR,
* the amount of DNA is measured after each cycle by the use of fluorescent markers that are incorporated into the PCR product. * The increase in fluorescent signal is directly proportional to the number of PCR product molecules (amplicons) generated in the exponential phase of the reaction. * Fluorescent reporters used include double-stranded DNA (dsDNA)- binding dyes, or dye molecules attached to PCR primers or probes that are incorporated into the product during amplification. * The change in fluorescence over the course of the reaction is measured by an instrument that combines thermal cycling with scanning capability. By plotting fluorescence against the cycle number, the real-time PCR instrument generates an amplification plot that represents the accumulation of product over the duration of the entire PCR reaction (Figure 1).
Figure 1—Amplification plots are created when the "fluorescent signal from each sample is plotted against cycle number; therefore, amplification plots represent the accumulation of product over the duration of the real-time PCR experiment....