Real Time-PCR

Prepared by:
Honorable GAKWAYA
Introduction
• Real time PCR (quantitative PCR) is polymerase chain reaction which
is fast, accurate and reliable technology that has revolutionized
molecular biology research.

• Real time PCR is advancement of the conventional PCR.

• Real time PCR amplifies, detect and quantifies DNA in real time
which does not require post PCR processing which saves the
resources and time,

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Introduction con’t
• It allows the accumulation of amplified product to be detected and
measured as the reaction progresses. That is in "real time".

• Detection of PCR products is based on the detection of fluorescent

produced by a reporter molecules which increases, as the reaction
proceeds. This occurs due to the accumulation of the PCR product
with each cycle of amplification.

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Introduction con’t

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Reaction components

• PCR master mix having appropriate fluorescent reporter

(i.e. dsDNA-binding dyes or probes)

• Sample (DNA or cDNA Template)

• Primers

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Basic principles

• During amplification, the fluorescent reporter binds into the

dsDNA within the target sequence.

• Binding of fluorescent dye/probe results in emission of

signal.

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Basic principles con’t

• Fluorescence emission increases as products accumulates.

• The increase in fluorescence emission during the PCR

reaction is then detected by fluorescent detectors in the
modified thermocycler.

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Basic principles con’t

• A computer software then constructs an amplification plots

using the fluorescence emission data that are collected
during the PCR amplification.

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Basic principles con’t

• CT is the basic principle

• CT cycle at which the products increase appreciably, hence the

fluorescent signals start to leave the background for detection and
quantification.

• It is determined mainly by the amount of template present at the start

of the amplification reaction.

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Basic principles con’t
• That is, large amount of template will require few

• That is, large amount of template will require few number of cycles to
accumulate enough products to give a fluorescent signal above background.
Similarly, a small amount of template will imply more.

• Thus, the reaction will have a high, or late, CT. This relationship forms the
basis for the quantitative aspect of real-time PCR.
Basic principles con’t
Detection of amplicons (PCR products)

• Two major types of fluorescent chemistries for amplicon detection;

1. DNA-binding dyes

example

• SYBR Green I,

• Ethidium bromide,

• EvaGreen
DNA-binding dyes (SYBR® green)

• DNA binding dye (binds dsDNA)

• Emits light when bound

• More double stranded DNA = more binding = more

fluorescence
SYBR® green
Advantages and disadvantages of
SYBR Green
• Advantages
Cheap

High sensitivity

• Disadvantages
Lack specificity (* Unspecific )
Detection of amplicons (PCR products) con’t

2. Fluorescent primer-and probe-based chemistries

These make use of Fluorescence Resonance Energy Transfer
(FRET) or some other form of fluorescence quencher, to
ensure that fluorescence is detected only in the presence of
amplicons.
• Include: TaqMan probe
TaqMan chemistry

• TaqMan probes are oligonucleotides that contain a fluorescent

dye (reporter), typically on the 5’base and a quenching dye
(quencher) typically located at 3’base.

• When irradiated, the excited fluorescent dye transfers energy to

the nearby quenching dye molecule rather than fluorescing,
resulting in non-fluorescent substrate.
TaqMan

• Reporter dye: Fluororescence: emit fluorescence to quencher

• Quencher dye: release heat energy
• Distance between reporter and quencher: 5base pairs
TaqMan principle
• During PCR, when the polymerase replicates/extends a template on
which a TaqMan probe is bound, the 5’exonuclease activity of the
polymerase cleaves the probe.

• This separates the fluorescent and quenching dyes and fluorescence

resonance energy transfer (FRET) no longer occurs.

• Fluorescence increases in each cycle in proportion to the rate of probe

cleavage.
Taqman
1) Denaturation and hybridization of
probe.

2) Extension of primer and strand

displacement of probe.

3) Cleavage of probe and fluorescence

from the reporter dye.
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REAL TIME PCR & IT’S FUNCTIONS IN DIAGNOSIS
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Extension
• Taq DNA polymerase digests the probe.
• ie. No transmission of fluorescence to
quencher because they are separated for a
long distance

• Release of fluorescence, but no release of

heat energy.
Advantages and disadvantages of Taqman

• Advantages of Taqman probes

• Very specific

• Disadvantages of Taqman probe

• Probes are expensive
How qPCR products are quantified?

• Methods used to quantify real-time PCR results,

1. Absolute quantification

2. Relative quantification

3. Melting curve analysis

Advantages of qPCR over conventional PCR

• Ability to monitor the progress of the PCR reaction as it occurs in real time

• Ability to precisely measure the amount of amplicons at each cycle, which

allows highly accurate quantification of the amount of starting material in
samples.

• Amplification and detection occurs in a single tube, eliminating post-PCR

manipulations
Quantitative real-time PCR (QPCR)

• QPCR is an important technique for quantifying messenger

RNA levels (gene expression) and DNA gene levels (copy
number) in biological samples

• It is one important application of Real-Time PCR.

Applications of real time PCR in diagnosis

• Quantitatively measurment of Human Immunodeficiency Virus (HIV).

• Detection of Thalassemia, hemophilia,Sickle cell anemia & favism.
• Phenyl ketonuria.
• Forensics for criminal cases
• Detection and Quantitation of Circulating Plasmodium falciparum DNA.
• Effect of antimicrobial peptides on host cells

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References

• Arya, M, Shergill, I.S., Williamson, M, Gommersall, L., Arya, N and Patel,

H.R.H. (2005). Basic principles of real-time quantitative PCR. Expert Rev.
Mol. Diagn. 5(2), 209-219.

• Real-time PCR handbook .(2012). Life Technologies Corporation.

Available from www.http:// lifetechnologies.com. Accessed on 28th

• Real-Time PCR Applications guide (2006). Bio-Rad Laboratories Inc.

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