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UNIVERSITY OF LAGOS

FACULTY OF SCIENCE

DEPARTMENT OF CELL BIOLOGY AND GENETICS

NAME: NLEMADIM UCHE TONY

MATRIC NUMBER: 150802006

TOPIC: REAL TIME PCR HIGH RESOLUTION ASSAY

SUPERVISOR: DR.O.O.IROANYA

DATE: 8 OF MARCH 2019


OUTLINE
INTRODUCTION
BASIC POLYMERASE CHAIN REACTION [PCR] COMPONENT
STEPS OF PCR
APPLICATION OF PCR
TYPES OF PCR
REAL TIME PCR
APPLICATION OF REAL TIME PCR
ROLES OF REAL TIME PCR
DIFFERENCES BETWEEN CONVENTIONAL PCR AND REAL TIME PCR
HIGH RESOLUTION MELTING ASSAY
APPLICATION OF REAL TIME PCR IN HIGH RESOLUTION MELTING
ASSAY [HRMA]
CONCLUSION.

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INTRODUCTION
The polymerase chain reaction [pcr] is a scientific
technique in molecular biology to amplify a single or a few
copies of dna across several orders of magnitude,generating
thousands to millions of copies of a particular dna sequence.
Kary Banks Mullis developed pcr in 1985 and was awarded
nobel prize in 1993.

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BASIC POLYMERASE
CHAIN REACTION
COMPONENT
Dna template (the sample dna that contains the target
sequence to amplify.
Deoxyribonucleosides triphosphates (dNTPs)
Pcr buffer,Mgcl2
Primers (forward and reverse)
DNA (Taq) polymerase
Pcr tubes
Thermal cycler

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STEPS OF PCR
There are three major steps in polymerase chain reaction
(pcr).
Denaturation (strand separation at 94 c to 96 c).
Annealing (primer binding at 45 c to 60 c)
Extension (synthesis of new dna at 72c)

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APPLICATION OF PCR
Medicine: Detecting infectious organism, discovering
variations and mutations in genes.
Genome project:Dna sequencing
The law:Genetic fingerprinting
Evolutionary biology:Taxonomic classification
Zoology:Research on animal behaviour

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TYPES OF PCR
Some of the common types of pcr are;
Real-time pcr
Conventional pcr
Nested pcr
Multiplex pcr
Arbitrary primed pcr
Hot-start pcr
Assembly pcr

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REAL TIME PCR
The real time polymerase chain reaction [pcr], also known
as quantitative real-time polymerase chain reaction [QRT-
PCR] or kinetic polymerase [KPCR], is a technique used to
simultaneously quantify and amplify a dna molecule. It is
used to determine whether a specific DNA sequence is
present in the sample and if it is present, the number of
copies in the sample.

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PRINCIPLES OF REAL
TIME PCR
In real-time Pcr, the real-time monitoring of the reaction is
achieved with the help of a fluorescent molecule added to the
pcr components.
This molecule exhibits a fluorescence signal that is
proportional to the amount of dna or rna amplified during the
reaction

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PRINCIPLE OF REAL
TIME PCR
Real-time pcr also makes use of special thermal cyclers
that can detect fluorescence and thus can monitor the
increase in fluorescence as the reaction progresses.

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DIFFERENCE BETWEEN
CONVENTIONAL PCR AND REAL TIME
PCR

COVENTIONAL PCR REAL TIME PCR


It is time consuming It is not time consuming
It collects data at the end-point of the It collects data during each cycle
reaction
It has very poor resolution (minimum It can detect very little changes
10 fold change) (minimum 2 fold change)
It uses agarose gel electrophoresis, It uses fluorescent dyes to detect the
to detect the amplified pcr product product

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HIGH RESOLUTION
MELTING ASSAY
 High resolution melting [HRM] analysis is a
new,simple,powerful and robust method for detecting dna
sequence variants.

 HRM analysis can discriminate dna sequence based on


their composition ,length ,or strand complementarity

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APPLICATION OF REAL TIME PCR IN HIGH
RESOLUTION MELTING ASSAY [HRMA]

 High resolution melting assay is performed on double stranded


dna samples.
 Typically the user will use the real-time polymerase chain
reaction prior to high resolution melting assay to amplify the dna
region in which their mutation of interest lies.

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APPLICATION OF REAL TIME PCR IN HIGH
RESOLUTION MELTING ASSAY [HRMA]
 Essentially the real time pcr process turns a tiny amount of your
region of dna of interest into a large amount so you have enough to
be worth analysing.
The region that is amplified is called amplicon.
After the pcr process,the HRMA begins
The process is simply a precise warming of the amplicon dna from
around 50c up to 95c,so the two strands of dna “melt” apart.

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CONCLUSION
Real time pcr amplifies the region of dna,for the detection of
dna sequence variance or mutant.

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THANK
YOU

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