REAL TIME PCR (RT-QPCR)

or QPCR

05-May-14 1
Outline

Introduction

1: Introduction Real Time PCR

2: Real Time PCR(RT-QPCR) Product Detection

3:Product Detection Applications

4:Applications Conclusion

5:Conclusion Reference
6:Reference

REAL TIME PCR (RT-QPCR) 05-May-14 2

Introduction to PCR
PCR was invented in 1984 by ( Kary mullis ) & he
received the Nobel Prize in chemistry in 1993, for Intro
his invention. duc
tion
Rea
It revolutionized biological methods specially in l Ti
me
molecular cloning in a way that it has PC
R
became an inseparable & irreplaceable part of Pro
Det duct
molecular investigations. ec t i
on
App
licat
ion
s
Con
clus
ion
Ref
e re n
ce

REAL TIME PCR (RT-QPCR) 05-May-14 3

more…
There is three basic steps which are in common
with all type of PCR Int
Thermal denaturation : rod
uct
In this step DNAs are denatured mostly by ion
Rea
temprature about 94˚c & single stranded DNAs are l Ti
me
PC
made. R
Pro
( in some cases It’s done by helicase ) Det duct
ec t i
Primer annealing : on
In this step Primers are attached to ssDNA by their App
l ic a
complementary regions. tion
s
Extension or polymerization : Con
clus
This is done by a temprature resistance polymerase ion
named Taq polymerase which is extracted from Ref
e re n
ce
Thermus aquaticus.

REAL TIME PCR (RT-QPCR) 05-May-14 4

Real Time PCR(RT-QPCR)
Int
ro duc
The perfect standard does not exist. tion
Rea
Quantitative Real-Time PCR is an important l Ti
me
technique for quantifying messenger RNA levels PC
Pro R
(gene expression) and DNA gene levels (copy Det duct
ec t i
number) in biological samples.(1) on
Additional benefits of Real-Time quantitative App
PCR include sensitivity and a wide dynamic l ic a
tion
range. As few as 10 copies of an RNA/DNA target s
can be detected with linearity of detection greater Con
clus
than six orders of magnitude. ion

Considered to be the most sensitive method for the Ref

e re n
ce
detection and quantification of gene expression

REAL TIME PCR (RT-QPCR) 05-May-14 5

 Real time PCR is the most accurate
method to detect:
Int
ro duc
Copy number of each gene Rea tion
l Ti
Amount of gene expression me
PC
Efficiency of drugs R
Virus infection Pro
Det duct
Different type of Pathogens ec t i
on
( CMV, streptococcus, mycobacterium,HIV & … App
lica
) tion
s
Methylation of DNA Con
clus
Different type of mutations ion
Adverse effect of organ transplantation Ref
e re nce

REAL TIME PCR (RT-QPCR) 05-May-14 6

 Detection in real time PCR

Uses fluorescence as a reporter by Three Int

general methods : ro
duc
tion
Rea
1. Hydrolysis probes l Ti
me
PC
R
(TaqMan, Beacons, Scorpions) Pro
2. Hybridization probes Det duct
e cti
on
(Light Cycler) most accurate & specific. App
licat
ion
3. DNA-binding agents s
Con
clus
(SYBR Green)less accuracy. ion
Ref
e re n
ce

REAL TIME PCR (RT-QPCR) 05-May-14 7

SYBR green ®

Int
* DNA binding dye ro duc
tion
* Binds to minor groove (dsDNA) Rea
l Tim
* Emits light when bound e PC
More double stranded DNA = more Pro R
binding = more fluorescence Det duct
ec t i
* Forensically, can be used to
on
calculate how much DNA was App
present before reaction. l ic a t
ion
s
* Unspecific Con
c lusi
on
Ref
er enc
e

REAL TIME PCR (RT-QPCR) 05-May-14 8

 Taqman

1) Denaturation and 1) Int

ro
hybridization of probe. duc
tion
Rea
l Tim
2) Extension of primer and e PC
R
strand displacement of Pro
probe. 2) Det duct
e cti
on
App
3) Cleavage of probe and licat
ion
fluorescence from the s
Con
reporter dye. clus
ion
Fluorescence from reporter dye is 3) Ref
e re n
directly proportional to the number ce
of amplicons generated

REAL TIME PCR (RT-QPCR) 05-May-14 9

 Molecular Beacons
MIDLAND is licensed by The Public Health Research Institute
of New York, Inc. to manufacture and sell Molecular Beacon
Probes. These probes, first described by Dr. Fred Russell Int
ro
duc
Kramer and his colleagues, make possible the in situ visual tion
detection of target DNA, and they also enable real-time PCR Rea
l Ti
quantitation. Use of different fluorophores coupled with careful me
PC
R
design of the probes permits distinguishing between sequences
Pro
differing by as little as one base. Det duct
e cti
on
App
licat
ion
s
Con
clus
ion
Ref
e re n
ce
A transmission and then the fluorescent images of a PtK2 cell inject with a ß-1
andrenergic mRNA MB at 3-minute intervals for 18 minutes.

REAL TIME PCR (RT-QPCR) 05-May-14 10

 Hybridization probes technique
Two oligo probes bearing a single dye each, one
with a fluorescein dye at the 3’ end and the other
Int
with a rhodamine dye at the 5’ end. When the rod
uct
ion
two oligos anneal to a complementary template, the Real
Tim
fluorescein dye is excited by the light source in the eP
CR
instrument and transfers its energy to the rhodamine Pro
D duc
dye via FRET. FRET can only occur when the two tec t e
tion
dyes are in close proximity.The instrument is set to
detect the rhodamime signal. App
lica
This results in the emission of fluorescence, which tion
s
subsequently can be detected during the annealing Con
clus
ion
phase and first part of the extension phase of the
Ref
PCR reaction. After each subsequent PCR cycle e re n
ce
more hybridization probes can anneal, resulting in
higher fluorescence signals.
REAL TIME PCR (RT-QPCR) 05-May-14 11
Real Time PCR Applications
*Quantitatively measurment of Human
Immunodeficiency Virus (HIV). Int
rod
*Detection of Thalassemia, hemophilia,Sickle cell uct
ion
Rea
anemia & favism by real time PCR. l Ti
me
*Cystic fibrosis. PC
R
*Phenyl ketonuria. Pro
Det duct
*Use in forensic medicine. ec t i
on
*Noninvasive Prenatal Diagnosis by Analysis of Fetal A
ppl
DNA in Maternal Plasma. ica
o n ti
*Detection and Quantitation of Circulating s
Con
Plasmodium falciparum DNA. clus
ion
*Effect of antimicrobial peptides on host cells
Ref
er enc
e

REAL TIME PCR (RT-QPCR) 05-May-14 12

 Conclusion

PCR has proved to be a useful tool in research and

diagnosis. However, its use has also brought new Introdu
c ti o
challenges to research. The sensitivity found in PCR Adv n
a
technology and the availability of quantitative disadvanntage
ta
results will bring new problems to the interpretation Real T ges
ime
of these results. A great deal of work is needed to P
Pro CR
generate a basis of knowledge for correct Det duct
ecti
interpretation of these tests. In medicine, PCR-based in on
med
diagnostics are just becoming widely used and i c in
e
because of the increased cost-effectiveness of the Conc
lusi
newer assays, knowledge for their interpretation will on
soon become available. Ref
e re nce

REAL TIME PCR & IT’S FUNCTIONS IN DIAGNOSIS 05/26/2021 13

 Reference
1) Real time PCR By M. Tevfik Dorak (Ed.). ISBN 0–203–96731–3 Master e-book
2) Clinical Applications of PCRSEECOND EDIITIION Edited by :Y. M. Dennis Lo,
Rossa W. K. Chiu & K. C. Allen Chan
3) JOURNAL OF CLINICAL MICROBIOLOGY, May 2005, p. 2435–2440
Int
4) Am. J. Trop. Med. Hyg., 75(2), 2006, pp. 212–218 rod
uct
Copyright © 2006 by The American Society of Tropical Medicine and Hygiene ion
5) Using Real Time RT-PCR in quantitatively measurment of HIV-1 A d
disa vanta
6) [Cooley's Anemia, Mediterranean Anemia. Includes: Thalassemia Major dv a ge
(Beta-Thalassemia Major), Thalassemia Intermedia,Thalassemia Minor (Beta-
nta g
Rea es
Thalassemia Minor, Heterozygous Beta-Thalassemia)] l Tim
7) Quantitative Real-Time PCR Assay for Rapid Identification of Deletion eP
Carriers in Hemophilia. Pro CR
8) Detection of unknown deleted genes in alpha-Thalassemia by real time pcr. Det duct
ecti
(Pasteur Institute of Iran) on
in m
9) Detection of cystic ®brosis alleles from single cells using molecular beacons and a edic
novel method of asymmetric real-time PCR
ine
10) Development of a Real-Time PCR Assay for Detection of Plasmodium Con
Falciparum clus
ion
11) Real-Time PCR for Detection and Identification of Plasmodium spp
Ref
http://www.oligos.com/MolecularBeaconProbes.htm er enc
e

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