This presentations will help you to understand a modification of PCR i.e. Real Time PCR. What are the components of a real time pcr and its methodology and its applications.

1. Real-Time PCR
(Quantitative PCR)
Name: Abhishek Anand Shetye.
Roll no.: 6001.
Subject: Biochemistry and Metabolism.

2. 1. Introduction
• Real time PCR, also known as Quantitative PCR (qPCR), is a
laboratory technique based on the Polymerase Chain Reaction
(PCR).
• Modification of PCR.
• Monitors amplification of targeted DNA molecule during the
PCR and not in the end of it.
• Can be used quantitatively and semi-quantitatively.

3. 2. Principle
• The principle behind qPCR is to monitor the accumulation of
PCR amplicons in “real time” by measuring the charge in
emission of fluorescence from either fluorescent DNA-binding
dyes or target-specific fluorescently labeled primers or probes
added to the PCR.

4. 3. Methodology
• In this method, the amount of DNA is measured after each cycle
with the use of either Fluorescent dyes or Probes.
• Increase in the fluorescence is directly proportional to the
PCR product generated.
• Fluorescent reporters used are:-
1. ds-DNA binding dyes.
2. Taq man probes / fluorophores.

5. Use of Fluorescent Dyes.
SyBr Green-
• A dye which only fluoresce when bound to ds-DNA.
• Change in the fluorescence is measured by an instrument which
combines thermal cycling with fluorescent dye scanning ability.
• An amplification plot is generated by the real-time PCR
instrument.

6. Plot of Fluorescence against the Cycle Number

7. Action of SyBr dye

8. Use of Probes
• TaqMan probe:
• Gene specific nucleic acid sequence joined to reporter and
quencher molecules.
• Probe binds in between two primers.
• Quencher and reporter are attached closely and when probe is
intact quencher absorbs the fluorescence of reporter molecule.
• During polymerization, Polymerase degrades the probe.
• Reporter and quencher are free, allowing Fluorescence.

9. Structure of Probe/Molecular Beacon
• Molecular beacon/ molecular probe consists 4 parts:
1. LOOP: 18-30 bp region complementary to the target sequence.
2. STEM: Lies on both ends of loop. 5-7bp long.
3. 5’ FLUOROPHORE: At 5’ end, attached a dye that fluoresces
in presence of a complimentary target.
4. 3’ QUENCHER: covalently attached at 3’ end and when
beacon is in closed loop shape, prevents fluorophore from
emitting fluorescence.

10. Diagrammatic structure of Molecular Probes

11. Applications
1. Ability to monitor the progress of the PCR reaction as it
occurs in real time and precisely measure the amount of
amplicon at each cycle.
2. Rapidly detect the nucleic acids of infectious diseases.
3. Microbial risk assessment of water quality and public
health protection.
4. Detection of phytopathogens.
5. Detection of GMOs.

12. References
• https://en.wikipedia.org/wiki/Real-
time_polymerase_chain_reaction#Basic_principles
• https://pubmed.ncbi.nlm.nih.gov/15833050/
• https://drive.google.com/file/d/1KWoEIiaEY_JbVw6klr
LiwEBT_vWc8pl2/view
• https://geneticeducation.co.in/real-time-pcr-principle-
procedure-advantages-limitations-and-applications/

13. Thank-You