Dr: Mohamed Maher

Research Assistant at National cancer

Institute (NCI)

Founder of Plasma Labs

Founder of plasma company

Phone : 01063729893

Mail : [email protected]
 Real time PCR

PCR = PCR, sometimes known as

“molecular photocopying’ : machine is
also known as thermal cyclers or
DNA amplifiers, or thermocyclers.
PCR machines amplify small
segments of DNA or RNA chosen
from the genome with a primer. It is
inexpensive and a very effective
tool.

PCR Machine- Principle, Parts, Steps, Types, Uses, Example and Data analysis
Principle of PCR
A single-stranded DNA template is used as a scaffold for the DNA polymerase enzyme, which guides the
synthesis of DNA from deoxynucleotide substrates. When a specially created oligonucleotide is annealed to a
longer template DNA. DNA polymerase adds nucleotides to the 3′ end of the molecule. Thus, DNA
polymerase can employ a synthetic oligonucleotide as a primer and extend its 3′ end to create an extended
stretch of double-stranded DNA when annealed to a single-stranded template with a region complementary to
the oligonucleotide.
 PCR Components (PCR reagents)

The five essential reagents include:

DNA template
DNA Polymerase
Primers
dNTPs(Deoxynucleotide triphosphate)
PCR Buffer
 DNA Template

It is crucial to choose the appropriate templates for PCR.

RNA templates are needed to create complementary DNA in RT-qPCR whereas DNA templates are needed
for traditional PCR.
Consequently, the first stage of a successful PCR requires the use of a reliable PCR template preparation kit.
 Oligonucleotide Primers

Primers are short strands of nucleotides (DNA or RNA) that are complementary to the
template DNA and act as a starting point for the DNA/RNA polymerase to begin synthesizing
new DNA.
Primers are short strands of nucleotides (DNA or RNA) that are complementary to the
template DNA and act as the DNA/RNA polymerase’s starting point for DNA synthesis. They
are necessary for the start of DNA synthesis. Lower temperatures (50–65°C) are needed for
annealing primers to single-strand DNA than for denaturation which generates hydrogen
bonds after the annealing process is finished.
 Thermostable DNA polymerase

Since the initial step of PCR requires separating DNA strands at a high temperature (90
°C), all PCR reactions require a DNA polymerase that can function at a high temperature
(about 70 °C).
A popular DNA polymerase for PCR known as Taq polymerase, obtained from the
thermophilic bacteria Thermus aquaticus is a heat-stable enzyme.
Deoxyribonucleotide triphosphate(dNTPs)
dNTPs are required for DNA polymerase to be able to synthesize
DNA.
 Buffer System

The best conditions are always maintained for the PCR reaction thanks to PCR buffers.
Tris-HCl, potassium chloride (KCl), and magnesium chloride (MgCl2) make up the majority of the
ingredients in the PCR buffer.
In order to keep the pH steady during PCR, tris-HCl and KCl are used. In order to ensure that DNA
polymerase performs properly DNA synthesis during PCR, magnesium ions serve as cofactors for the
enzyme.
Steps of PCR
1- Denaturation
When the reaction mixture is heated for 0.5 to 2 minutes to 94°C, denaturation takes place.
A single-stranded DNA is created as a result of the hydrogen bonds between the DNA’s two
strands being broken.
The single DNA strands now serve as a template for the synthesis of additional DNA strands.
2- Annealing
For around 20 to 40 seconds, the reaction temperature is decreased to 54 to 60 °C.
The primers attach to the template DNA’s complimentary sequences in this circumstance.
Primer sequences are 20–30 bases long, single-strand DNA or RNA segments.
They act as the precursor in the production of DNA.
There are two primers—a forward primer and a reverse primer—the two separated strands run in opposing
directions.
3- Elongation
The temperature is increased to between 72 and 80 degrees Celsius at this stage.
The Taq polymerase enzyme tacks the bases onto the primer’s 3′ end. As a result, the DNA stretches from 5′ to 3′.
Taq Polymerase can withstand extremely high temperatures. A double-stranded DNA molecule is produced as a
result.
In order to obtain several DNA sequences of interest in a short amount of time, these three procedures are done
20–40 times.
DATA analysis

Amplification plots

PCR analysis

Standard curve

Sample analysis
Amplification plot
 Phases of real time pcr

4- End point Detection

( 26-38)
Reaction has stopped and no more product
are being made and if long enough pcr
product will being degrade

Reaction component are being consumed

Reaction is slowing and product are starting to
degrade

2-Amplification of product every cycle

1- Stationary baseline phase ( 16 – 25 )
Initiation phase ( 0-15)
Noise or background signals
 The cycle threshold value ( CT)

Number of the cycles needed to amplify the nucleic acid to detectable level
 CT Value

Exact amount of DNA

Relative amount of DNA
 CT of sample 1 = 17
CT of sample 2 = 19

Sample 2 = 4 amplification sample 1

)2N )
Calibration curve .. Stander curve
1. Conventional PCR machine
The conventional polymerase chain reaction is used to
amplify a target DNA sequence to several million in a
short amount of time, usually in just 2-3 hours. It allows
the replication of cellular genetic material using a
polymerase enzyme to construct specific fragments of
DNA.
The polymerase enzyme works alongside a primer which
is connected to a strand of DNA, allowing for the synthesis
of specific parts of the DNA strand. The result of using
primers in this way is the amplification of a chosen DNA
sequence, up to millions or billions of copies.
Conventional PCR is used in many areas of study,
including medical and diagnostic research, forensic
studies and research, selective DNA isolation,
amplification and quantification of DNA.
2-qPCR
Quantitative PCR (qPCR), also called real-time PCR, or
RT-PCR, is a variation of the standard polymerase chain
reaction which uses just one machine to combine the
amplification of a target DNA sequence with the
quantification of the concentration of the DNA in any given
reaction. This is done using fluorescence-detecting
thermocyclers.
Compared to conventional PCR, qPCR provides a faster
alternative to facilitate analysis by detecting products in
real-time during the exponential phase. Fluorescent dyes
signal DNA of interest, and the amount of fluorescence
generated is determined by the quantity of DNA present.
There are various models of real-time PCR available, but
they all have common features: a standard thermal cycler
platform coupled with an excitation source (usually a laser
or tungsten lamp), a camera for fluorescence detection,
and computer and software for data processing.
qPCR can be used in genotyping and quantification of
pathogens, microRNA analysis, cancer detection,
3. Reverse Transcription
Reverse Transcription PCR (RT-PCR) is a variant on
conventional polymerase chain reaction which amplifies
target RNA. The addition of reverse transcriptase (RT)
enzyme before PCR means it is possible to amplify and
detect RNA targets.
During RT-PCR, RNA molecules are converted into
complementary DNA (cDNA). Single-stranded cDNA is
converted into double-stranded DNA using DNA
polymerase. The resulting DNA molecules can then be
used and amplified in a PCR reaction.
RT-PCR is used in gene insertion, research methods,
genetic disease diagnosis and cancer detection.
4-Nested
Nested PCR is a modification of PCR where non-specific
binding is prevented to increase the sensitivity and
specificity of the reaction. Nested PCR works by having
the first set of primer bind to the outside of the target DNA
and amplifies a larger fragment, while a second set of
primer binds specifically at the target site in successive
PCR reactions.
Nested PCR is great for use in phylogenetic studies and in
the detection of different pathogens. This is due to the
higher sensitivity brought by Nested PCR compared to
conventional PCR. Even if a sample contains lower DNA,
Nested PCR allows this sample to be amplified.
 5-Hot Start
Hot start PCR is a new form of the conventional
polymerase chain reaction which reduces the occurrence
of undesired products and formation of primer-dimers due
to non-specific DNA amplification at room temperatures.
This works by keeping the different elements of the
reaction separate until the mixture reaches the
denaturation temperature after heating.
Hot start PCR can often increase product yields in
comparison to conventional PCR. It also requires less
effort than conventional PCR and reduces the risk of
contamination.
6-Digital (dPCR)
Digital PCR devices, or dPCR, are the most accurate
devices on the market. They provide absolute counts of
target DNA with enhanced increased sensitivity, precision,
and reproducibility. Digital polymerase chain reaction is
poised to disrupt molecular analysis technology on every
level. There are two types of dPCR machines:
Droplet Digital PCR (ddPCR): ddPCR uses Taq
polymerase to amplify targeted DNA in a complex sample.
To simplify the processing, ddPCR emulsifies samples in
oil and uses fluorescence to process and analyze the
results. Before analysis, the sample is divided into
droplets and thermocycled, then run through a 96 well
PCR plate. This separation process is the primary
difference between ddPCR and qPCR
qdPCR: The qdPCR process is based on integrated
fluidic circuits (chips). While chip-based techniques have a
narrower dynamic range, they provide extremely precise
sample partitioning and vastly lower
 Genomic DNA Extraction and Genomic DNA Isolation

Methods for genomic DNA isolation

Three general methods for genomic DNA isolation are common, and each is based on a different
biochemical principle. Selection of a method is based on the throughput required, equipment available in
the lab, and how the purified genomic DNA will be used.

Organic extraction and precipitation

Genomic DNA isolation by organic extraction involves the addition of phenol and quanidine isothiocynate
to separate the DNA and proteins into different organic phases. Organic extraction is a low-cost method
and, with advanced reagents such as DNAzol, is a straightforward method requiring very little equipment.

Silica
DNA binds to silica (aka glass fibers) under high-salt conditions and can be released under low-salt
conditions. Silica-containing columns provide an easy way to bind, wash, and elute purified genomic DNA
from multiple clarified cell lysates in parallel.
Columns are designed to flow buffers through centrifugation, vacuum, or gravity. PureLink Genomic DNA
Mini Kits use spin column technology and work in a microcentrifuge for easy prep of up to 18 samples at a
time. Spin plates provide the same isolation technology in a high-throughput, automation-friendly format.

Paramagnetic beads
In this method, paramagnetic (attracted to magnet) beads are added to the sample, and genomic DNA
binds to the beads. Using a strong magnet, the beads are held in place while removing unwanted
material. After washing, the genomic DNA is eluted from the beads in water or a low-salt buffer. The bead-
based method, used in MagMAX multi-sample DNA isolation and related kits, is scalable and automation
compatible.