Real time PCR allows for monitoring of DNA amplification during polymerase chain reaction (PCR), rather than just at the end. There are two main detection methods: using non-specific fluorescent dyes that bind to double stranded DNA, and using sequence-specific fluorescent probes. Common non-specific dyes include SYBR Green I, while TaqMan probes are an example of sequence-specific probes that use fluorescence resonance energy transfer. Real time PCR has applications in disease diagnosis, microbiology research on food and water safety, and quantifying gene expression levels.

1. Real Time PCR
Naren Yadav

2.  What is Real time PCR ?
 A real-time polymerase chain reaction 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, and not at its end, as in
conventional PCR.
 qPCR

3. 2 common detection methods
in Real time PCR :-
 (1) By using non-specific fluorescent dyes.
 (2) By using sequence-specific fluorescent
probes.

4. 1) Non-specific fluorescent
dye

5.  dsDNA binding dyes :-
 SYBR green I
 SYBR green II
 EVA green
 LC green
 YO-PRO
 SYTO family

6. (SYBR green I):-

7. Instrumentation of real time
PCR :

8. Observation :-

9. SYBR green fluorescence chart produced in real-time PC
Threshold
Baseline
CT

10.  Baseline – the initial cycles of PCR when the
fluorescent signal shows no or little change.
 CT value – Thresold cycle (CT) indicates the
cycle number where the reaction fluorescence
crosses the thresold. This reflects the point in
reaction when sufficient amplicons have been
generated to give significant fluorescent signal
over the baseline.
 Standard curve – a curve consisting of CT
values plotted against the log of standard
concentration. The concentration of unknown
samples are extrapolated from the standard

11. Melting curve produced at the end of real-time
PCR.

12. 2) Sequence specific
Fluorescent probe method

13. General principle :-
o Based on sequence specific oligonucleotide
probes.
o Fluorogenic (reporter dye) at 5’ end and
quencher dye at 3’ end.
o Hybridizes to the target gene either in
between the primers or as a part of one of the
oligonucleotide primer.
o FRET (Fluorescence Resonance Energy Transfer)

14. TaqMan probe :-

15. Haip pin probes - Scorpions :-

16. Applications of Real time PCR
:-
 Diagnostic uses – To diagnose infectious
diseases
 Microbiological uses - Used by microbiologists
in the fields of food safety, food spoilage and
fermentation and for the microbial risk
assessment of water quality.
 Uses in research - used to provide quantitative
measurements of gene transcription.

17. • Thank you