Topic Name: Applications of Genetic

Engineering
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Unit 4

Lecture 33: Real Time PCR

Standard PCR vs Real Time PCR

▪ standard PCR is used to detect the presence or absence of certain genomic

fragments, whereas Real-Time PCR is used to detect the expression level
of that fragment in the organism. Amount of template added affects
amount of product detected after a fixed number of cycles and this can
be used as a way of measuring the quantity of target DNA in the original
sample.
▪ standard PCR is more time consuming, as it uses agarose gel
electrophoresis to detect the amplified PCR products. Whereas, Real Time
PCR is less time consuming as it can detect the amplifications during the
run after each cycle.
▪ standard PCR collects data at the end-point of the reaction, where as Real
Time PCR collects data during the exponential phase.

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Standard PCR

▪ standard PCR has very poor resolution(minimum 10 fold

change) while real-time PCR can detect very little
changes(minimum 2 fold change) due to the high resolution.
▪ standard PCR uses Ethidium Bromide and UV light to
visualize bands in the agarose gel medium, while Real Time
PCR uses fluorescent dyes to detect the product.
▪ To know how many cycles are required to produce detectable
amount of product:
 Set up number of amplification reactions and stop after
different number of cycles and see which reaction gives
detectable product- Tedious and lengthy procedure
 A way of detecting the product without interrupting the series
of amplification-Real Time PCR

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Real Time PCR
▪ the concept of monitoring DNA amplification in real time
through monitoring of fluorescence
▪ It monitors the amplification of a targeted DNA molecule
during the PCR, i.e. in real-time, and not at its end, as in
standard PCR. Real-time PCR can be used quantitatively
(quantitative real-time PCR), and semi-quantitatively, i.e.
above/below a certain amount of DNA molecules (semi
quantitative real-time PCR).
▪ In real time PCR (also denoted as quantitative PCR—qPCR;,
fluorescence is measured after each cycle and the intensity of
the fluorescent signal reflects the momentary amount of DNA
amplicons in the sample at that specific time

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Q PCR
• In initial cycles the fluorescence is too low to be
distinguishable from the background. However, the point at
which the fluorescence intensity increases above the
detectable level corresponds proportionally to the initial
number of template DNA molecules in the sample. This point
is called the quantification cycle (Cq; different manufactures of
qPCR instruments use their own terminology, but since 2009,
the term Cq is used exclusively) and allows determination of
the absolute quantity of target DNA in the sample according to
a calibration curve constructed of serially diluted standard
samples (usually decimal dilutions) with known
concentrations or copy numbers (Yang and Rothman, 2004;
Kubista et al., 2006; Bustin et al., 2009).

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Steps of Q PCR

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Real Time PCR-measuring gene expression by mRNA quantification

• SYBR green emits fluorescence in

the presence of ds DNA.
• As the PCR proceeds the amount of
product(dsDNA) increases and
hence the amount of fluorescence
increases.
• Linear relationship between X
threshold cycle (Ct) at which
fluorescence begins to increase
rapidly and the number of template
molecules in the starting sample.
• Ct value can be used to calculate
the concentration of template in the
original sample.
• As the amount of template is
reduced the Ct value increases in a
linear fashion

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This molecular tool is well-suited for the rapid detection of bacteria directly in clinical specimens,
allowing early, sensitive and specific laboratory confirmation of related diseases. It is particularly
suitable for the diagnosis of infections caused by fastidious growth species, and the number of these
pathogens has increased recently. This method also allows a rapid assessment of the presence of
antibiotic resistance genes or gene mutations
With the increasing amount of sequencing data available, it is literally possible to design qPCR
assays for every microorganism (groups and subgroups of microorganisms, etc.) of interest. The main
advantages of qPCR are that it provides fast and high-throughput detection and quantification of
target DNA sequences in different matrices. The lower time of amplification is facilitated by the
simultaneous amplification and visualization of newly formed DNA amplicons. Moreover, qPCR is
safer in terms of avoiding cross contaminations because no further manipulation with samples is
required after the amplification. Other advantages of qPCR include a wide dynamic range for
quantification (7–8 Log10) and the multiplexing of amplification of several targets into a single reaction
(Klein, 2002). The multiplexing option is essential for detection and quantification in diagnostic qPCR
assays that rely on the inclusion of internal amplification controls (Yang and Rothman, 2004; Kubista
et al., 2006; Bustin et al., 2009).
qPCR (with the inclusion of reverse transcription for the diagnostics of RNA viruses) provides the
appropriate sensitivity and specificity (Hoffmann et al., 2009). Moreover, determination of the viral
load by (RT)-qPCR is used as an indicator of the response to antiviral therapies

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Steps of Q PCR

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Threshold line is created by the instrument. That fluorescene signal
which can be detected by the instrument

Ct=1/initial copy no. of

template
Application
Relative quantification by Realtime PCR

Q. You have 1000 cancerous cells and 1000 normal cells, compare the expression of
p53 gene in these two cell types.
Sample-cancer cells
Control-normal cells
Thank You