Real time polymerase chain reaction

1. CHHAVI KAUSHAL
M.Sc Forensic Science &
Criminology 4th
semester
REAL TIME POLYMERASE
CHAIN REACTION

2. Contents
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Introduction
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History
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Role of RT-PCR
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Real time PCR with double
standard DNA binding dyes
as reporters
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Fluorescent Reported Probes
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Two-step qPCR
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One-step qPCR
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Working
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Types of real time PCR
markers
●
Advantage over normal
PCR
●
Applications in Forensics
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References

3. Introduction
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It is a method which screens the
amplification of targeted DNA
molecule during PCR in real time.
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This monitoring of DNA molecule
during real time makes it different
from conventional PCR.
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It can be used qualitatively,
quantitatively or semi
quantitatively.

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Quantitative real time PCR is also known as qPCR.
Detection of PCR products in real time PCR is done by
using two methods:
1) Non-specific fluorescent dyes intercalating with double
stranded DNA.
2) Sequence specific DNA probes containing
oligonucleotides labelled with fluorescent reporter.
Fluorescent reporter helps in detection after hybridization
of probe with its complementary sequence.

5. Continue...
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In real time PCR, the sample is illuminated with a beam of
light of a particular wavelength to detect fluorescence
emitted by the excited fluorophore.
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Heating and cooling of samples is performed by thermal
cycler.
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A series of temperature changes takes place in PCR
process which is repeated 25-50 times.
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The technique used can be either non-specific
fluorochromes or hybridization probes.

6. History
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1993 – First real-time PCR detection experiments to show utility for
DNA quantization reaction took place using EtBr detection
(illumination,CCD camera detection).
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1996 – TaqMan detection methods used, instead of EtBr, for real-
time detection of PCR.
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1996-7 – ABI introduces first real-time qPCR (“Sequence Detection
System”) instrument (the ABI 7700).
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Since then – many more instrument manufacturers (Roche,
BioRad, Stratagene, Corbett, Cepheid and MJ) and many more
detection methods have been developed.

7. Role of Real Time PCR
Beside normal amplification process performed by normal PCR, Real
Time PCR can perform detection, analysis and quantification of the
sample.
●
Detection: Find out the presence of targeted gene sequence which is
assured by the presence of the amplification curve.
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Quantification: Quantification of targeted DNA in a sample can be done
by using the cycle no. needed to obtain the threshold value of detector
and PCR efficiency.
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Analysis: Analysis of the variants can be done by studying the melting
curve or comparing the melting temperature with the sequences of the
database.

8. Real-Time PCR with Double-Stranded
DNA-Binding Dyes as Reporters
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Double stranded DNA in PCR binds to the DNA binding dye which causes
fluorescence.
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Fluorescence intensity at each cycle increases with increase in DNA product
during PCR. The dye binds to all the PCR products including non-specific PCR
products. This binding prevents accurate monitoring of intended target
sequence.
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The same preparation of reaction is done with real time PCR along with
addition of fluorescent double stranded DNA dyes. The intensity of the
fluorescence is measured after each cycle of real time PCR.
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The fluorescence can be seen only if the dye binds to the double stranded
DNA which is the PCR product. A pair of primers is needed to carry out the
amplification reaction in real time PCR.

9. Fluorescent Reported Probes
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Complementary DNA sequence to the probe is detected by using
fluorescent reporter probes.
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Specificity significantly increases in reporter probe. This technique
can be performed in case of other double stranded DNA as well.
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Monitoring of several target sequences can be done in the same
tube using differently colored labels, fluorescent probes in
multiplex assay.
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The interference caused by primer dimers is also avoided due to
specificity of this technique.

10. Continue...
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The method depends on a DNA-based probe with a fluorescent reporter at one end
and a quencher of fluorescence at the opposite end of the probe.
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Detection of fluorescence cannot take place in case of close proximity of quencher
to the reporter.
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5’ to 3’ exonuclease activity of the Taq polymerase breaks the probe which leads to
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breakdown of reporter-quencher proximity.
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This breakdown allows unquenched emission of fluorescence. This fluorescence
can be detected after excitation with a laser.
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A proportionate rise in fluorescence is produced due to rise in the targeted product
by the reporter probe at each PCR cycle.
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This proportionate rise is due to the breakdown of the probe and release of the
reporter.

12. Two-step qRT-PCR
Two type of instrumentation is available for real time PCR:
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Two-step quantitative reverse transcriptase PCR (qRT-PCR) starts with the
reverse transcription of either total RNA or poly(A)+ RNA into cDNA using a
reverse transcriptase (RT).This first-strand cDNA synthesis reaction can be
primed using random primers, oligo (dT), or gene-specific primers (GSPs).
To give an equal representation of all targets inreal-time PCR applications
and to avoid the 3 bias ofoligo(dT) primers, many researchers use random
primersor a mixture of oligo(dT) and random primers.The temperature used
for cDNA synthesis depends on theRT enzyme chosen. Next,
approximately 10% of the cDNAis transferred to a separate tube for the
real-time PCRreaction.

13. One-step qRT-PCR
One-step qRT-PCR combines the first-strand cDNA
synthesis reaction and real-time PCR reaction in the
same tube, simplifying reaction setup and reducing the
possibility of contamination. Gene-specific primers
(GSP)are required. This is because using oligo(dT) or
random primers will generate nonspecific products in
the one-step procedure and reduce the amount of
product of interest.

14. Working
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The working procedure of Real Time PCR can be divided in two
steps:
Amplification: (Same like normal PCR)
1. Denaturation:
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High temperature incubation is used to "melt” double- stranded
DNA into single strands and loosen secondary structure in single-
stranded DNA.The highest temperature that the DNA polymerase
can withstand is typically used (usually 95°C). The denaturation
time can be increased if template GC content is high.

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2. Annealing: During annealing, complementary sequences
have an opportunity to hybridize, so an appropriate
temperature is used that is based on the calculated melting
temperature (Tm) of the primers(5°C below the Tm of the
primer).
3. Extension: At 70-72°C, the activity of the DNA
polymerase is optimal, and primer extension occurs at rates
of up to 100 bases per second. When an amplicon in real-
time PCR is small, this step is often combined with the
annealing step using 60°C as the temperature.

16. Detection:
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The detection is based on fluorescence technology. The specimen is
first kept in proper well and subjected to thermal cycle like normal
PCR but at this machine it is subjected to tungsten or halogen source
that lead to fluoresce the marker added to the sample and the signal
is amplified with the amplification of copy number of sample DNA.
The emitted signal is detected by an detector and sent to computer
after conversion into digital signal that is displayed on screen.
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The signal can be detected when it comes up the threshold
level(lower detection level of detector), this cause elimination of
background noise.

19. Types of Real Time PCR Markers
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Taqman probe (Hydrolysis probe)
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SYBR Green
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Hairpin probes (Molecular Beacons)
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Scorpion Primers
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Hybridization Probes
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Sunrise Uniprimer Probe

21. Types of Real Time PCR Markers
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There are many different markers used as the marker of Real Time PCR.
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There are mainly two types of marker are used for this purpose.
1.Taqman probe.
2.SYBR Green.
Taqman Probe:
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This is a hydrolysis probe, it bear a reporter dye , often fluorescein(FAM)
at its 5’ end and a quencher tetramethylrhodamine (TAMRA), attached to
the 3’ end of a oligonucleotide .In normal condition the probe remain
coiled on itself bringing the fluorescence dye near the quencher causing
quenching of fluorescent signal of the dye.

22. Continue...
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The oligonucleotide of the Taqpolymerase has a homologous s region with the target
gene, when the target sequence is present in mixture it bind with the sample DNA.
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As the taqpolymerase start to replicate new DNA strand at the extension stage it
causes degradation of the probe by 5’ end nuclease activity and the fluorescein is get
separated from the quencher and fluorescence signal is generated.
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As this procedure continue at each cycle the no. of signal molecule increase causing
the increase of signal which is positively related with the amplification of DNA.

25. SYBR Green:
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This is a dye that provide prominent fluorescent signal when
bind at the minor groove of DNA nonspecifically. Other
fluorescent dyes like Ethidium Bromide or Acridine Orange
can also be used but SYBR Green is better used for its
higher signal intensity.
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SYBR Green is better then the Taqman Probe as it can
provide the information about each cycle as well as about the
melting temperature that is not obtained by Taqman probe.
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But it suffer for the disadvantage of no specificity which is
assured by Taqman Probe so SYBR Green can suffer from
signaling for unexpected DNA materials.

30. Advantage over Normal PCR
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It does not require gel preparation like traditional PCR.
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It is not time consuming like normal PCR.
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Less complexity at the quantification of sample.etc.

31. Applications in Forensics
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There are various applications of PCR in the field of genetic analysis. PCR can be used
for pathogen identification, mutation analysis, template quantitation, gene
deletion analysis, high throughput SNP genotyping, linkage analysis, diet
analysis, RNA detection and even in forensic analysis.
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To discriminate one person from entire world population genetic fingerprinting is used.
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Isolation of minute samples of DNA from crime scene is compared with suspect’s
sample or DNA database.
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PCR helps in the amplification of DNA to give conclusive results.
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DNA fingerprinting is also used for paternity testing in which an individual is matched
with their close relatives.
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Testing of DNA from unknown human remains is done and then linked with parents,
siblings or any close relative.

32. References
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https://www.sciencedirect.com/topics/agricultural-and-bi
ological-sciences/real-time-polymerase-chain-reaction
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https://www.slideshare.net/pratyayseth/real-time-pcr-34
159486
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https://www.frontiersin.org/articles/10.3389/fmicb.2017.
00108/full
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Biotechnology- Spanding Horizons (B. D. Singh)

33. Thankyou