real time presentation.pptx molecular biology

Real-time PCR: Principle, Procedure, Advantages, Limitations and Applications

1. A  real-time polymerase chain reaction  ( real-time PCR ) , also known as  quantitative polymerase chain reaction  ( qPCR ), is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR (i.e., in real time), not at its end, as in conventional PCR.

2. SYBR/CYBR Green Chemistry

3. Taqman Chemistry

4. Threshold cycle(Ct) The  Ct  (cycle threshold) is defined as the number of cycles required for the fluorescent signal to cross the threshold ( ie exceeds background level ). The threshold line  is the level of detection or the point at which a reaction reaches a fluorescent intensity above background levels.

5. Threshold cycle(Ct) C q   (Ct) values are inverse to the amount of target nucleic acid that is in the sample , and correlate to the number of target copies in the sample. Lower C q  values (typically below 29 cycles) indicate high amounts of the target sequence. Higher C q  values (above 38 cycles) mean lower amounts of the target nucleic acid. 

6. Amplification plots 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.

7. Absolute quantification Absolute quantitation uses serially diluted standards of known concentrations to generate a standard curve. The standard curve produces a linear relationship between C t  and initial amounts of total RNA or cDNA, allowing the determination of the concentration of unknowns based on their C t  values . This method assumes all standards and samples have approximately equal amplification efficiencies 

8. Absolute quantification

9. Relative quantification

10. Melting curve Melting curve analysis is an assessment of the dissociation characteristics of double-stranded DNA during heating. As the temperature is raised, the double strand begins to dissociate

11. A successful real-time PCR experiment will have the following characteristics: