qPCR
Quantitative Polymerase Chain Reaction
Lecture by:
Yepy Hardi R
This section will discuss the foundations of
quantitative PCR technology
INTRODUCTION
Quantitative PCR
Commonly known as Real Time PCR or Kinetic PCR
Based on polymerase chain reaction (PCR) principle
Amplify and simultaneously quantify a targeted DNA
molecule
It enables both detection and quantification of a specific
sequence in a DNA sample.
The amplified DNA is quantified as it accumulates in the
reaction in real time after each amplification cycle.
Two common methods of quantification:
fluorescent dyes that intercalatewith double-strand DNA
modified DNA oligonucleotide probes that fluoresce when
hybridized with a complementary DNA.
http://www.ga-
international.com/PCR%20LABEL.100_0879CROP.jpg
Expression Quantification
Expression level of a gene can be estimated by
some other method such as northern blotting or
microarray
Gene amplification by PCR Detect gene
expression at minute levels from single/small
number of cells
For mRNA-based PCR the RNA sample first needs
to be reverse transcribed to cDNA via the
enzyme, reverse transcriptase.
The amplified product is measured at the end of
each PCR cycle using Fluorophores
The data can be analyzed by computer software
to calculate relative gene expression between
several samples, or mRNA copy number based on
a standard curve.
http://www.altabioscience.bham.ac.uk/services/oligo/beacons.shtml
How is DNA detected during PCR process?
Detection method
Double-Stranded DNA Dyes
Binds to all double-stranded (ds)DNA
fluorescence of the dye.
An increase in DNA product increase in
fluorescence intensity
Also binds to nonspecific PCR products
(e.g. primer dimers).
The reaction = PCR + fluorescent dsDNAdye.
With reference to a standard dilution, the dsDNA
concentration in the PCR can be determined.
http://www.gene-quantification.de/chemistry.html
The fluorescence
measurement is
performed during
extension step.
Fluorescent Reporter Probe
The most accurate and most reliable
(but also the most expensive)
It uses a sequence-specific RNA or
DNA-based probe to quantify only the
DNA containing the probe sequence
Significantly increases specificity, and
allows quantification even in the
presence of some non-specific DNA
amplification.
Allows for multiplexing by using
specific probes with different-coloured
labels
http://www.shinegene.org.cn/image/image004.jpg
Fluorescent Reporter Probe
Taqman Probe
TaqMan probes are oligonucleotides that have a fluorescent reporter dye attached to the 5' end
and a quencher moiety coupled to the 3' end.
These probes are designed to hybridize to an internal region of a PCR product.
In the unhybridized state, the proximity of the Fluor and the quench molecules prevents the
detection of fluorescent signal from the probe.
During PCR, when the polymerase replicates a template on which a TaqMan probe is bound, the
5'- nuclease activity of the polymerase cleaves the probe.
This decouples the fluorescent and quenching dyes and FRET no longer occurs. Thus,
fluorescence increases in each cycle, proportional to the amount of probe cleavage
http://en.wikipedia.org/wiki/Image:TaqMan_Probes.jpg
Fluorescent Reporter Probe
Molecular Beacons
Designed to remain intact during the amplification
reaction, and must rebind to target in every cycle for
signal measurement.
Form a stem-loop structure when free in solution.
prevents the probe from fluorescing.
When a Molecular Beacon hybridizes to a target, the
fluorescent dye and quencher are separatedthe
fluorescent dye emits light upon irradiation.
Can be used for multiplex assays by using spectrally
separated fluor/quench moieties on each probe.
2) Renaturation
(Capturing signal)
The Benefit
Only 2.5 3 hours
overall
no additional time for
gel electrophoresis
and staining-destaining
Time
efficiency
Time
efficiency
only add small
amount of SYBR
Green dye.
no agarose gel and
DNA markers
Reaction cost
efficiency
Reaction cost
efficiency No hazardous
component
No Ethidium
Bromide needed
Safe Safe
On the spot
interpretation for
quick observation of
the sample.
Quick
Observation
Quick
Observation
Two strategies are commonly employed to quantify the results obtained
by real-time RT-PCR;
the standard curve method
the comparative threshold method
Quantitation of Results
Standard Curve
First constructed from an RNA of known concentration First constructed from an RNA of known concentration
Used as a reference standard for extrapolating
quantitative information for mRNA targets of unknown
concentrations.
Used as a reference standard for extrapolating
quantitative information for mRNA targets of unknown
concentrations.
RNA stability can be a source of variability RNA stability can be a source of variability
It would involve the construction of cDNA plasmids in vitro
transcribed into the RNA standards accurately quantitated
a time-consuming process.
It would involve the construction of cDNA plasmids in vitro
transcribed into the RNA standards accurately quantitated
a time-consuming process.
The use of absolutely quantitated RNA standards will help
generate absolute copy number data.
The use of absolutely quantitated RNA standards will help
generate absolute copy number data.
Standard Curve
Other nucleic acid samples can be used to construct the standard
curve (e.g. purified plasmid dsDNA, in vitro generated ssDNA or
any cDNA sample expressing the target gene)
Other nucleic acid samples can be used to construct the standard
curve (e.g. purified plasmid dsDNA, in vitro generated ssDNA or
any cDNA sample expressing the target gene)
Spectrophotometric measurements at 260 nm can be used to assess the
concentration of these DNAs, which can then be converted to a copy
number value based on the molecular weight of the sample used.
Spectrophotometric measurements at 260 nm can be used to assess the
concentration of these DNAs, which can then be converted to a copy
number value based on the molecular weight of the sample used.
cDNA plasmids are the preferred standards for standard
curve quantitation.
cDNA plasmids are the preferred standards for standard
curve quantitation.
This method will only yield information on relative
changes in mRNA expression.
This method will only yield information on relative
changes in mRNA expression.
This, and variation introduced due to variable RNA inputs,
can be corrected by normalization to a housekeeping
gene.
This, and variation introduced due to variable RNA inputs,
can be corrected by normalization to a housekeeping
gene.
Amplification Curve
4.397x10
7
copy/rx; Ct1: 19
4.397x10
5
copy/rx; Ct2: 23
4.397x10
4
copy/rx; Ct3: 27
4.397x10
3
copy/rx; Ct4: 30
4.397x10
2
copy/rx; Ct5: 34
Sample WSSV; Ctx: 24
???? Copy/rx
NTC emerge ??
Primer Dimer ????
Standard Curve
4.397x10
7
copy/rx;
Ct1: 19
4.397x10
5
copy/rx; Ct2: 23
4.397x10
4
copy/rx; Ct3: 27
4.397x10
3
copy/rx; Ct4: 30
4.397x10
2
copy/rx; Ct5: 34
Sample wssv; Ct5: 24
Internal Control
Primer
Dimer
Melt Curve
Amplicon
Primer dimer
Thank you
