Translated version of Real-Time PCR.

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PCR

AUTOMATIC ANALYSIS - D. ALI IBRAHIM

5 - Real-Time PCR
This interaction also called quantitative real time polymerase chain
reaction Quantitative Q-PCR / QPCR Or kinetic polymerase chain reaction :
A laboratory technique Depend On the interaction of the PCR To
amplify a specific DNA molecule at the same time Reveal For this
molecule .
Is used to detect qualitative and quantitative DNA molecule to a
specific .

The principle technical :

Her The same principle of the PCR Ordinary But where they feature a
specific DNA inflation and reveal the DNA amplifier output and
measure the quantity in each session ( In other words, real-time In
real time , Ie, each session of the interaction ).
It is more sensitive and less time consuming than
the PCR reaction Normal because it does not need to analyze the
outputs in the final point Of interaction Such as the use of gel
electrophoresis to investigate the amplification products .
In The past few years Started to become the PCR From the traditional
to the Real-Time .

Comparison between the PCR And Real-Time PCR :

Can detect the amplified output of the Real-Time PCR And interaction
in the first outburst ( Exponential phase ) Unlike the methods
of PCR Traditional product, which was investigating the amplification
in a way to gel electrophoresis and interaction developed in the final
or final in his point .

The interaction of the defects PCR In the final point :

Some of the problems or difficulties experienced in detecting the

final point :
1 - Bad tuning .
2 - Low sensitivity of the detection method in the investigation of
the amplification product by gel Unlike the real-time .
3 - The ability of discrimination and segregation in a weak gel
requires a difference in the concentration of 10 Times, unlike
the Real-time Which requires a different amount of double .
4 - Is an automated .
5 - Discrimination based on size only .
6 - No results can be expressed as numbers .
7 - The use of bromine Alathediom crayon is insufficient
quantitative .
8 - Be treated after the end of the PCR reaction .
9 - Need for a very long time . It could take several days .
As you can see in the following figure found in the gel samples
containing 10 Copy and other 50 Copy .

Difficult to distinguish between the two samples Thoian DNA there is

a difference between them amounted 5 Agarose gel-fold . While Realtime Unable to distinguish between my sample DNA there is a
difference between the two-fold ( Any 10 Copies for 20 Copy ).

Phases of the PCR :

To understand the reasons for the fact that the PCR Must be limited
traditional understanding of what happens during this interaction .
Can Tks J M the process of interaction of the PCR Basic to 3 Phases :
Exponential phase : Occurs in this phase of the product has
doubled in each d Orh imposing that the effective

interaction 100% , Interaction in this phase is very specific and

controlled .
Linear phase ( Significant difference ): Components of the
interaction starts running out and become a slow speed .

Developed a
threshold ( The final point ): Detected on the gel for the
traditional methods ): Interaction depends not make new copies of
the product and if you leave the amplification products for a long
time, these products begin Baltkhrb .

Common methods for the interaction of the Real Time PCR :

1 - Almtflorh use dyes that interfere with the dual-stranded DNA .
2 - Probes of DNA composed of several modified nucleotides,
which shines when Taatahjn with Taq DNA corresponding CMOS .

Astkh Long as it came with ^ Aliyy E Almtf Orh :

The link between the dye is in the Bthelm small double-stranded
DNA leads to the brilliance of the dye .
Therefore, the increased concentration of double-stranded DNA
in the PCR reaction Will lead to an increase in fluorescence
intensity or fluorescence measured at each PCR cycle ( At the end
of elongation stage because all of the DNA molecules are in this
stage, two-stranded ) Which allows for monitoring the increasing
amounts of the PCR product From cycle to another .
Vsbgh dye such as SYBR Green Will be linked with any non-specific
double-stranded DNA molecule, even with molecules of
the PCR Non-specific ( Even with the molecules of the prefixes
associated with each result and the complementarity between
Nukluotadadtha, constitute the so-called Primer dimers ) Which
preventsMeasurement The exact To the amount of DNA Target .
Interaction being a cyclist in the thermal Thermocycler The
measured fluorescence intensity at the end of each session using
a detector or reader where fluoridation or shine Taatflor dye only
when combined with dual-stranded DNA ( Any output of the PCR ).

Road E Almsabi T pregnant E level of Orh :

Is the most accurate method and reliability, but more expensive .
It uses probes made from DNA or RNA quality of the output to be
detected, so the quality of this method where possible to measure
the concentration of target DNA, even in the event of non-specific
products .
To Several forms of this method :

1 - Method TaqMan method :

This method is usually performed using

a probe of DNA or RNA carries one end end 5 Article Almtflorh In
the second 3 Article suppressor or ballasts of fluoridation .
Very close between the holder and brake prevents the
fluorescence detection of fluorescence . The destruction of the
probe byTaq enzyme Polymerase from the end 5 To the end 3 Lead
to the spacing holder and brake fluorescence or phase in the
process of elongation, which allows the measurement of
fluorescence or fluorescence of the holder of the fluorination . The
more a result of amplification in each PCR cycle Will increase the
intensity of fluorescence is a result of damage the probe direction
and the emancipation of pregnant fluorination .
1 - The PCR reaction handsome As is customary in
the PCR reaction Normal with the addition of the probe carrier of the
fluoridation .

2 - Once the interaction begins, and in every stage of the annealing

of primers and probe target Sttahjn to Almrsaf .
3 - Will create a new DNA strand from the primers and the
polymerase enzyme up as soon as the probe to probe it
Sikhrb ( Physically separated from the brake carrier )Which leads to
increased fluorescence .
4 - Fluorescence detected and measured in a toddler warming and
that are commensurate with the amount of output amplifier .

2 - Another form for the use of hybridization probes :

Add Msparin Noaaan cascade of nucleotides present in the
central region of the target DNA template .
One marked by probes in the end 3 Bafilorucein while the second
probe in the end, marked by 5 Another example, a red dye Red
640 dye . Nucleotide sequence of both rovers is chosen so that the
rovers hybridization with a piece of DNA arranged head
amplifier - Tail and thus to bring Abbgtin very close to each other .
Filorucein Athaj by light from the source of a toddler warming
and issued light Mtflor green wavelength slightly longer than the
wavelength of light absorbed, and when Abbgtin Qreptan too
each other, the energy from the Filorucein excite red pigment
related to the probe II and thus made light of fluoridation red
color along the longer wave . This transfer of energy is referred to
as FRET Transfer of energy from Filorucein to red pigment, which
depends on the distance between Dzeita Abbgtin . Transmitted
power is effective only if the distance between the Abbgtin by 15 Nucleotides only .
Fluorescence intensity measured in this way in the process of
annealing .
Match the intensity of fluorescence in direction with the
increasing amount of DNA amplified .

 Show fluorescence signal only when both rovers Atahjn with

single strand DNA in the process of annealing, while it does not
happen in the process of metamerism .

3 - Molecular Beacons :
Probes a few nucleotides are issued when Taatahjn shine with a
target sequence of nucleotides was due to DNA or RNA molecule .
These probes where the structure of secondary structure such as
hair tongs result of the integration of some of the internal
nucleotides with each other and at the end of the endings Mtflorh
pigment dye in the other inhibitory . As a result of secondary
structure ( Hair tongs ) The Abbgtin Tkonan Qrbetin each of which
prevents the formation of glittering brilliance of the dye .
However, these probes are subject to change when a formal
Taatahjn with Almrsaf which leads to a spacing between Abbgtin
and thus the emergence of glittering brilliance of the dye in the
process of annealing proportional to the amount of singlestranded Almrsaf formed .
This method allows Palmairat multi-purpose interaction of
different genes and one using probes linked to the quality, but
with a different color dyes Mtflorh .

Applications of Real-time PCR :

You can use this technique in routine applications for
the PCR reaction The traditional . As well as for applications that do
not fit with the PCR The traditional :
1 - Quantitative calibrations nevi .
2 - Quantification of gene expression .
3 - The effectiveness of drug treatment .
4 - Measurement of DNA damage .
5 - In the discovery of infectious pathogens .
6 - Genetic profiling .
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Department of Laboratory Diagnosis - first year