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