Real-time PCR theory and...
Transcript of Real-time PCR theory and...
Real-time PCR theory and applications
By Dalia M. Sabri, PhD
Lab Manager , BRC, SCU
Biotechnology Research Center, Suez Canal University, 11-11-2015
* PCR History
* What is PCR?
* Endpoint PCR Vs Real-time PCR
* Real-time PCR
* QPCR Detection Chemistry
* Real-time PCR Instruments
* Methods of Quantification
* New technologies
PCR History 1985 PCR first published
Late1980’s PCR used for quantification, but required electrophoresis to detect product at
end-point of PCR
1991 Use of 5’-> 3’ exonuclease activity of Taq polymerase to detect specific PCR
activity (“TaqMan” approach).
1992 Discovery that EtBr (dsDNA probe) can be added to the PCR mix and that the
fluorescence of EtBr will increase at each cycle of PCR due to product formation.
No need to cool to room temperature or to open tubes to measure the increased
EtBr fluorescence. First real-time experiments – in closed PCR tubes on UV-
trans-illuminator. Higuchi and others at Roche and Chiron .
1993 First real-time PCR detection experiments to show utility for DNA quantitation.
Improved EtBr detection (illumination, CCD camera detection). Higuchi and
others at Roche .
1996 TaqMan detection methods used, instead of EtBr, for real-time detection of PCR.
Improved specificity. Heid and others at ABI and Genentec.
1996-7 ABI introduces first real-time qPCR (“Sequence Detection System”) instrument
(the ABI 7700).
Since then Many more instrument manufacturers (Roche, BioRad, Stratagene, Corbett,
Cepheid, MJ) and many more detection chemistries.
What is PCR?
* The Polymerase Chain Reaction (PCR) is a revolutionary technology
developed by Kary Mullis in the 1980s. PCR is based on using the
ability of DNA polymerase to synthesize new strand of DNA
complementary to the offered template strand, that results in generating
thousands to millions of copies of a particular DNA sequence from a
single copy or a few copies of a piece of DNA .
*The method relies on thermal cycling, consisting
of cycles of repeated heating and cooling of the
reaction for DNA melting and enzmatic replication
of the DNA.
The three-step PCR process
1. Denaturation step (Separating the Target DNA)
DNA template is heated to more than 90 ºC, which separates the double-
stranded DNA into two separate single strands.
The three-step PCR process
2. Annealing step (Binding Primers to the DNA Sequence)
The reaction is cooled between 40 and 60 ºC. PCR oligonucleotides bind
(anneal) to sequences on either side of the target DNA region, marking off
the sequence for step three
The three-step PCR process
3. Extension step (Making a Copy)
The temperature is increased to 72 ºC, nucleotides in the solution are added to the
annealed primers by the DNA polymerase to create a new strand of DNA
complementary to each of the single template strands. After completing the
extension, two identical copies of the original DNA have been made.
PCR amplification curve
• Exponential
- Exact doubling of product is accumulating at every cycle.
- The reaction is very specific and precise.
- All of the reagents are fresh and available, the kinetics of the reaction push the reaction to favor doubling of amplicon.
• Linear (high variability)
- As the reaction progresses, some of the reagents are being consumed in amplification process.
- The reactions start to slow down and the PCR product is no longer being doubled at each cycle.
• Plateau (End-point)
-The reaction has stopped, no more products are being made and if left long enough, the PCR products will begin to degrade.
Endpoint PCR Vs Real-time PCR
Endpoint PCR Vs Real-time PCR
Endpoint PCR Real-time PCR
Measures the amount of
accumulated PCR product at the
end of the PCR cycles
Measures PCR amplification as it
occurs
Reaction endpoint
data collection
Ct
Endpoint PCR Vs Real-time PCR
Endpoint PCR Real-time PCR
Semi-quantitative
Though comparing the intensity of
the amplified band on a gel to standards of a known
concentration.
Quantitative
Data collection during the exponential (log) phase makes the
quantity of the PCR product is directly proportional to the amount
of template nucleic acid.
Endpoint PCR Vs Real-time PCR
Endpoint PCR Real-time PCR
Semi-quantitative
Though comparing the intensity of
the amplified band on a gel to standards of a known
concentration.
Quantitative
Data collection during the exponential (log) phase makes the
quantity of the PCR product is directly proportional to the amount
of template nucleic acid.
Endpoint PCR Vs Real-time PCR
Endpoint PCR Real-time PCR
Applications
Amplification of DNA for:
Sequencing
Genotyping
Cloning
Applications
Gene expression quantification
Microarray verification
Quality control and assay validation
Pathogen detection
SNP genotyping
Copy number variation
MicroRNA Analysis
Viral quantification
Endpoint PCR Vs Real-time PCR
Endpoint PCR Real-time PCR
Disadvantages:
- Low sensitivity.
- Post-PCR processing.
- Low resolution and Poor Precision.
-Non-automated.
-Size-based discrimination only &
results are not expressed as numbers.
-Ethidium bromide for staining is not very
quantitative.
Advantages:
- Increased dynamic range of detection
- No post PCR processing.
- Detection is capable down to a 2-fold
change.
- Collects data in the exponential growth
phase of PCR.
-An increase in reporter fluorescent signal is
directly proportional to the number of
amplicons generated.
-The cleaved probe provides a record for
amplified amplicons.
Real-time PCR
Real-time PCR *Real-time PCR combines PCR amplification and detection into a
single step.
*With Real-time PCR, fluorescent dyes are used to label PCR
products during thermal cycling with the capacity to illuminate
each sample with a beam of light of at least one specified
wavelength .
*Real-time PCR instruments measure the accumulation of
fluorescent signal emitted by the excited fluorophore.
QPCR Detection Chemistry
1- Non-specific detection (DNA binding dyes): SYBR Green, BEBO, BOXTO, EvaGreen.
2- Specific detection (Probe based test): TaqMan, Molecular Beacons, Hybridization Probes (Scorpions, Amplifluor Primers, LUX Primers, FRET probe pairs).
SYBR Green I SYBR Green I dye binds to double-
stranded DNA and emits fluorescence
only when bound, DNA-dye-complex
absorbs blue light (λ= 497 nm) and emits
green light (λ=520 nm). The stain can also
binds to single-stranded DNA and RNA
but with lower performance.
SYBR Green I SYBR Green I dye binds to double-
stranded DNA and emits fluorescence
only when bound, DNA-dye-complex
absorbs blue light (λ= 497 nm) and emits
green light (λ=520 nm). The stain can also
binds to single-stranded DNA and RNA
but with lower performance.
*Advantages:
1- Low cost assay.
2-Easy to design and set up.
*Disadvantages:
1-Non specific system as it detects all ds
DNA , in poorly designed PCR reaction
primer-dimer product will be detected
and quantified
2-Not adapted to multiplex .
TaqMan probe-based assay TaqMan probes consist of a fluorophore
reporter attached to the 5’-end of
the oligonucleotide probe and a quencher at the
3’-end. The quencher molecule quenches the
fluorescence emitted by the fluorophore when
excited by the cycler’s light source.
TaqMan probe-based assay TaqMan probes consist of a fluorophore
reporter attached to the 5’-end of
the oligonucleotide probe and a quencher at the
3’-end. The quencher molecule quenches the
fluorescence emitted by the fluorophore when
excited by the cycler’s light source.
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TaqMan probe-based assay TaqMan probes consist of a fluorophore
reporter attached to the 5’-end of
the oligonucleotide probe and a quencher at the
3’-end. The quencher molecule quenches the
fluorescence emitted by the fluorophore when
excited by the cycler’s light source.
Advantages:
1- very specific
2- Can be used in multiplex qPCR assays
Disadvantages:
1- difficult to design
2- More expensive than SYBR Green
assay
Applications: *Gene expression assays
*Determine the viral load in clinical specimens
*Bacterial Identification assays
*SNP genotyping
*Verification of microarray results
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Real-time PCR Instruments
Any Real-time PCR Instrument consists of two main components
Any Real-time PCR Instrument consists of two main components
Thermal Cycler
(PCR machine)
Optical module ( to detect fluorescence in
the tubes during the run)
1- Halogen lamp
2- Filter /detector :
(5 dyes detectors)
FAM/SYBR Green I, VIC/JOE,
NED/TAMRA/Cy3,
ROX/Texas Red, Cy5
3- CCD camera
Any Real-time PCR Instrument consists of two main components
Methods of Quantification Absolute Quantification Relative Quantification
Quantification of unknown samples based on a known quantity. First you create a standard curve; then you compare unknowns to the standard curve and extrapolate a value.
This method analyze changes in gene expression in a given sample relative to another reference sample (such as an untreated control sample).
Ct (cycle threshold) is defined as the number of cycles required
for the fluorescent signal to cross the threshold (exceeds
background level).
Threshold is the point of detection.
Cycle-Threshold (Ct) cycle at which sample crosses threshold
Rn
Cycle
Melting curve: The melting curve analysis is based on the Tm, in which half of the DNA molecules are
in the form of single strand and the other half in the form of a double helix.
The Tm is dependent on DNA composition, since each amplified fragment has a specific
size and base composition, they can be identified by melting curve analysis.
FIGURE : Melting curve analysis for HBV genotyping.
A (68 bp), D (119 bp) and F (97 bp)
Ref. : BECKER, Carlos Eduardo et al . MELTING CURVE ANALYSIS FOR THE SCREENING OF HEPATITIS B VIRUS GENOTYPES A, D AND
F IN PATIENTS FROM A GENERAL HOSPITAL IN SOUTHERN BRAZIL. Arq. Gastroenterol., São Paulo , v. 50, n. 3, p. 219-225, Sept. 2013 .
Why we should collect results during exponential phase?
Plateau effect
Rn
Exponential Phase
Real-time PCR Applications
New technologies
Digital PCR Digital PCR works by partitioning a sample into many individual real-
time PCR reactions; some portion of these reactions contain the target
molecule (positive) while others do not (negative). Following PCR
analysis, the fraction of negative answers is used to generate an absolute
answer for the exact number of target molecules in the sample, without
reference to standards or endogenous controls.
Digital PCR QX100™ Droplet Digital
Bio-Rad
(A)
Samples partitioning
into 20,000 nanoliter-
sized droplets
(B)
PCR on a thermal cycler
(C)
The detector reads each
droplet on turn at the
rate 1000 droplet per sec
• No need to rely on references or standards
• Ability to increase precision by using more PCR replicates
• High tolerance to inhibitors
• Capability to analyze complex mixtures
• Linear detection of small-fold changes
Digital PCR Advantages
Digital PCR Applications
• Copy number variation with unrivaled accuracy.
• Rare sequence detection with unmatched sensitivity.
• Mutation detection.
• Gene expression analysis with the highest precision.
• miRNA analysis.
• Next generation sequencing sample quantification.
PCR is a tool
It is your decision how to use it!!?