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Real time PCR

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.

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Real Time PCR Naren Yadav
 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
2 common detection methods in Real time PCR :-  (1) By using non-specific fluorescent dyes.  (2) By using sequence-specific fluorescent probes.
1) Non-specific fluorescent dye
 dsDNA binding dyes :-  SYBR green I  SYBR green II  EVA green  LC green  YO-PRO  SYTO family
(SYBR green I):-
Instrumentation of real time PCR :
Observation :-
SYBR green fluorescence chart produced in real-time PC Threshold Baseline CT
 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
Melting curve produced at the end of real-time PCR.
2) Sequence specific Fluorescent probe method
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)
TaqMan probe :-
Haip pin probes - Scorpions :-
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.
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