Real-Time PCR

1. RT-PCR
Real Time PCR
Course Instructor: Ji-Seon Jeong
Presenter: Mesele Tilahun Belete
Place : KRIBB; Republic of Korea
University of Science and Technology,
Korean Research Institute of Bioscience and Biotechnology

2. What is real time PCR?
-is the continuous collection of fluorescent signal from one or more polymerase chain
reactions over a range of cycles.
-called quantitative real-time PCR, is a technique used to amplify and simultaneously
quantify a targeted DNA molecule. It enables both detection and quantification

3. Real Time PCR/ SpectroFluorometer

4. Real time PCR
 relies on all the components of standard PCR
detect any dsDNA in the medium

5. Real Time PCR non-specific Detection
• The cyber green detect the minor group of the DNA and at each cycle we
will get more fluorescent b/c more cyber green bind to the DNA

6. Real Time PCR specific Detection (TaqMan)

10. Real time PCR
 Most sensitive and quantitative approach to PCR
 Detection of PCR product accumulated in the reaction
 Real time PCR relies on all the components of standard PCR
 Additionally it uses fluorescent oligo nucleotide probe
 Probe produce a fluorescent signal in each time a double standard product is formed
 Probe help to monitor the reaction in real time
 Probe contains 2 fluorescent dyes
1. Reporter dye on 5’end
2. quencher dye on 3’end
 When the reporter is excited by light it transfer its to quencher. This energy transfer is called fluorescence reson
ance energy transfer [FRET]
 It prevents the reporter dye from meeting the light
 During annealing step, probe bind to the template DNA
 Annealing temperature for probe is 5-10oc
 As the temperature lowered primer come and anneal the DNA
 Taq-polymerase synthesize new DNA by adding base to the primer
 Taq-DNA polymerase have 5’-3’ exo nuclease activity that cleave the terminal nucleotides of ds DNA
 Exonuclease remove the nucleotides from template DNA
 Probe under go degradation. It allows the detection of fluorescent dye
 Fluorescent signal is produced in each time a ds DNA product is formed
(171) The principle of Real Time PCR, Reverse Transcription, quantitative rt-PCR - YouTube