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151298 DE DIOS Juan Carlos P. Bi 140.

1 A 15 May 2018

The main difference in end-point and real-time PCR is the monitoring of amplification
using DNA binding fluorescent dyes in real-time PCR which provides quantitative data of the
amount of DNA or mRNA converted to cDNA. Real-time allows for the detection of PCR
amplification during the early phases of the reaction. This provides a distinct advantage over
end-point PCR detection. End-point uses agarose gels for detection of PCR amplification at the
final phase or end-point of the PCR reaction.
Real-time PCR gives relative quantitative expression on the basis of detection of DNA by
a fluorescent dye (i.e. SYBR green) in real time unlike conventional PCRs wherein the results
can be interpreted at the end of the PCR reaction.
Real-time PCR is a more sensitive and accurate method for analyzing the quantitative &
qualitative expression of particular genes at different set of experimental conditions or in
different tissues or organisms. Conventional PCR gives qualitative expression of the presence
of the gene.
In Real-time PCR, the quality of the run/amplification can be monitored during the run by
analyzing the melt curve and Ct values for controls and test samples whereas in end-point PCR
the results can only be detected at the end of the reaction after the PCR products are loaded
onto an Agarose gel with EtBr and visualized under UV light.
Real time PCR is less time consuming and labor intensive as compared to end-point
PCR. End-point detection is in general very time consuming. Results may not be obtained for
days and are based on size discrimination, which can be unreliable. End-point resolution is very
poor compared to Real-Time PCR which can detect as little as a two-fold change.
Some of the problems with End-Point Detection:
Poor Precision
Low sensitivity
Short dynamic range < 2 logs
Low resolution
Non - Automated
Size-based discrimination only
Results are not expressed as numbers
Ethidium bromide for staining is not very quantitative
Post PCR processing

Advantages of using Real-Time PCR


Traditional PCR is measured at end-point (plateau), while real-time PCR collects data in the
exponential growth phase
Increase in Reporter fluorescent signal is directly proportional to the number of amplicons
generated
The cleaved probe provides a permanent record amplification of an Amplicon
Increase dynamic range of detection
No-post PCR processing
Detection is capable down to a 2-fold change
Real-Time PCR Applications ​(these are brought upon mostly by real-time’s ability to collect
data in the exponential growth phase)
Viral Quantitation
Quantitation of Gene Expression
Array Verification
Drug Therapy Efficacy
DNA Damage measurement
Quality Control and Assay Validation
Pathogen detection
Genotyping

References:

Perkel JM. 2015 Oct 29. dPCR vs. qPCR vs. Endpoint PCR. Biocompare. [accessed 2018 May
14].
Available from
https://www.biocompare.com/Editorial-Articles/179491-dPCR-vs-qPCR-vs-Endpoint-PCR/

Real-Time PCR vs Traditional PCR. Applied Biosystems [Internet]. [accessed 2018 May 13].
Available from http://www6.appliedbiosystems.com/support/tutorials/pdf/rtper_vs_tradper.pdf