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8/3/2019 Copy of Real Time PCR
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Real-time Polymerase Chain Reaction
Real-time polymerase chain reaction, also called quantitative real time polymerase
chain reaction (Q-PCR/qPCR/qrt-PCR) or kinetic polymerase chain reaction (KPCR), is a
laboratory technique based on the PCR , which is used to amplify and simultaneously quantify atargeted DNA molecule. For one or more specific sequences in a DNA sample, Real Time-PCR
enables both detection and quantification. The quantity can be either an absolute number of
copies or a relative amount when normalized to DNA input or additional normalizing genes.
Background:
Various techniques i.e.;
1. Differential display2. RNAse protection assay
3. Northern blotting
Were used for the detection and quantification of the of the gene expression.
• These methods were not suitable because they require large amount of DNA or RNA.
• Northern blotting technique only provides qualitative or semi-quantitative information of
mRNA level.
• So the scientists were in a need to robustly detect and quantify gene expression from
small amount of RNA.
• For this purpose amplification of gene transcript is necessary.
• Simple PCR can amplify the gene but the quantity can only be detected at the end of the
reaction.
• To check the quantity at any any run time PCR is modified by the use of PCR and highly
sensitive monitor connected with the computer which we called REAL TIME PCR.
Procedure:
General principle as followed by PCR, but following common methods are used to detect the
products in Real Time PCR.
1. TaqMan probe (sequence specific probe).
2. Molecular Beacon (sequence specific probe).
3. Scorpion primer/probe (primer specific probe).
4. DNA binding agent (SYBR Green dye).
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TaqMan probe:
The Taqman probe. The red circle represents the quenching dye that disrupts the
observable signal from the reporter dye (green circle) when it is within a short distance.
The probe consists of two types of fluorophores, which are the fluorescent parts of
reporter proteins (Green Fluorescent Protein (GFP) has an often-used fluorophore). While the
probe is attached or unattached to the template DNA and before the polymerase acts, the
quencher (Q) fluorophore (usually a long-wavelength colored dye, such as red) reduces the
fluorescence from the reporter (R) fluorophore (usually a short-wavelength colored dye, such as
green). It does this by the use of Fluorescence (or Förster) Resonance Energy Transfer (FRET),
which is the inhibition of one dye caused by another without emission of a proton. The reporter
dye is found on the 5’ end of the probe and the quencher at the 3’ end.
The TaqMan probe binds to the target DNA, and the primer binds as well. Because the
primer is bound, Taq polymerase can now create a complementary strand.
Once the TaqMan probe has bound to its specific piece of the template DNA after
denaturation (high temperature) and the reaction cools, the primers anneal to the DNA. Taq
polymerase then adds nucleotides and removes the Taqman probe from the template DNA. This
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separates the quencher from the reporter, and allows the reporter to give off and it emits its
energy. This is then quantified using a computer. The more times the denaturing and annealing
takes place, the more opportunities there are for the Taqman probe to bind and, in turn, the more
emitted light is detected.
The reporter dye is released from the extending double-stranded DNA created by the Taq
polymerase. Away from the quenching dye, the light emitted from the reporter dye in an excited
state.
Molecular beacons:
Molecular beacons is short segments of single-stranded DNA. The sequence of each
molecular beacon must be customized to detect the PCR product of interest.
Attached to opposite ends of the beacon are a fluorescent reporter dye and a quencher
dye. When the molecular beacon is in the hairpin conformation, any fluorescence emitted by the
reporter is absorbed by the quencher dye and no fluorescence is detected.
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Molecular beacon is 33 nucleotides long with a reporter dye attached to the 5' end and a
quencher attached to the 3' end. The nine 5' bases are able to form base pairs with the nine 3'
bases which bring the reporter and quencher in very close proximity. Therefore, when the
reporter is excited by the appropriate light, its emission is absorbed by the quencher and no
fluorescence is detected. The pink lines represent nucleotides that can form base pairs with the
PCR product under investigation.
The PCR portion of real-time PCR is standard. Two PCR primers are used to amplify asegment of DNA (Figure 2).
The two primers are show as purple arrows and the base pairing between the two strands are
shown in pink.
As the PCR continues, the newly synthesized PCR products are denatured by high
temperatures. As each strand of the product are separated, the molecular beacon also is denatured
so the hairpin structure is disrupted. As the temperatures cool for the next round of primer
annealing, the molecular beacon is capable of forming base pairs with the appropriate strand of
the PCR product. Any molecular beacons that do not bind to PCR product reform the hairpin
structures and thus are unable to fluoresce. However, molecular beacons that bind to PCR
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product remove the ability for the quencher to block fluorescence from the reporter dye.
Therefore, as PCR product accumulates, there is a linear increase in fluorescence.
Detection of PCR product by molecular beacon. When the beacon binds to the PCR product, it is
able to fluoresce when excited by the appropriate wavelength of light. The amount of
fluorescence is directly proportional to the amount of PCR product amplified.
Real-time PCR can be performed in a "multiplex" format which means that more than
one PCR product can be detected in a single reaction tube. For each sequence, there is a unique
color of fluorescent dye and therefore, each PCR product is associated with its own color which
is detected by the real-time PCR machine.
Scorpion primer/probes:
Scorpion primer/probes, sequence-specific priming and PCR product detection is
achieved using a single oligonucleotide. The Scorpion probe maintains a stem-loop configuration
in the unhybridized state. The fluorophore is attached to the 5' end and is quenched by a moiety
coupled to the 3' end. The 3' portion of the stem also contains sequence that is complementary to
the extension product of the primer. This sequence is linked to the 5' end of a specific primer via
a non-amplifiable monomer. After extension of the Scorpion primer, the specific probe sequence
is able to bind to its complement within the extended amplicon thus opening up the hairpin loop.
This prevents the fluorescence from being quenched and a signal is observed.
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SYBR Green Dye
Applied Biosystems has developed conditions that permit the use of the SYBR Green dyein PCR without PCR inhibition and increased sensitivity of detection compared to ethidium
bromide.
How the SYBR Green Dye Works:
The SYBR Green dye uses the SYBR Green dye to detect polymerase chain reaction (PCR)
products by binding to double-stranded DNA formed during PCR. Here’s how it works:
• When SYBR Green I dye is added to a sample, it immediately binds to all double-
stranded DNA present in the sample.
• During the PCR, DNA Polymerase amplifies the target sequence, which creates the PCR
products, or "amplicons."
• The SYBR Green dye then binds to each new copy of double-stranded DNA.
• As the PCR progresses, more amplicons are created. Since the SYBR Green dye binds to
all double-stranded DNA, the result is an increase in fluorescence intensity proportionate
to the amount of PCR product produced.
Advantages of SYBR Green Dye:
• It can be used to monitor the amplification of any double-stranded DNA sequence
• No probe is required, which reduces assay setup and running costs
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Disadvantage of SYBR Green Dye:
• The primary disadvantage of the SYBR Green I dye chemistry is that it may generate
false positive signals; i.e., because the SYBR Green I dye binds to any double-stranded
DNA, it can also bind to nonspecific double-stranded DNA sequences.
Quantification:
Quantifying gene expression by traditional DNA detection methods is unreliable.
Detection of mRNA on a Northern blot or PCR products on a gel or Southern blot does not allow precise quantification. For example, over the 20-40 cycles of a typical PCR, the amount of DNA
product reaches a plateau that is not directly correlated with the amount of target DNA in the
initial PCR.
Real-time PCR can be used to quantify nucleic acids by two methods: relative
quantification and absolute quantification. Relative quantification is based on internal reference
genes to determine fold-differences in expression of the target gene. Absolute quantification
gives the exact number of target DNA molecules by comparison with DNA standards.
The general principle of DNA quantification by real-time PCR relies on plotting
fluorescence against the number of cycles on a logarithmic scale. A threshold for detection of
DNA-based fluorescence is set slightly above background. The number of cycles at which the
fluorescence exceeds the threshold is called the cycle threshold, Ct. During the exponential
amplification phase, the sequence of the DNA target doubles every cycle. For example, a DNA
sample who’s Ct precedes that of another sample by 3 cycles contained 2 3 = 8 times more
template. However, the efficiency of amplification is often variable among primers and
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templates. Therefore, the efficiency of a primer-template combination is assessed in a titration
experiment with serial dilutions of DNA template to create a standard curve of the change in Ct
with each dilution. The slope of the linear regression is then used to determine the efficiency of
amplification, which is 100% if a dilution of 1:2 results in a C t difference of 1.
To quantify gene expression, the Ct for an RNA or DNA from the gene of interest is
divided by Ct of RNA/DNA from a housekeeping gene in the same sample to normalize for
variation in the amount and quality of RNA between different samples. This normalization
procedure is commonly called the ΔΔC t -method and permits comparison of expression of a gene
of interest among different samples. However, for such comparison, expression of the
normalizing reference gene needs to be very similar across all the samples. Choosing a reference
gene fulfilling this criterion is therefore of high importance, and often challenging, because only
very few genes show equal levels of expression across a range of different conditions or tissues.
Mechanism-based qPCR quantification methods have also been suggested, and have the
advantage that they do not require a standard curve for quantification. Methods such as MAK2
have been shown to have equal or better quantitative performance to standard curve methods.
These mechanism-based methods use knowledge about the polymerase amplification process to
generate estimates of the original sample concentration.
Advantages of using Real-Time PCR:
• Traditional PCR is measured at end-point (plateau), while real-time PCR collects data in
the exponential growth phase
• An increase in reporter fluorescent signal is directly proportional to the number of
amplicons generated
• The cleaved probe provides a permanent record amplification of an amplicon
•Increased dynamic range of detection
• Requirement of 1000-fold less RNA than conventional assays
• No-post PCR processing due to closed system (no electrophoretical separation of
amplified DNA)
• Detection is capable down to a 2-fold change
• Small amplicon size results in increased amplification efficiency (even with degraded
DNA)
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Real-Time PCR Applications:
Real-Time PCR can be applied to traditional PCR applications as well as new
applications that would have been less effective with traditional PCR. With the ability to
collect data in the exponential growth phase, the power of PCR has been expanded into
applications such as:
• Copy number variation (CNV)
• Quantitation of gene expression including NK cell KIR gene expression
• Array verification
• Biosafety and genetic stability testing
• Drug therapy efficacy / drug monitoring
• Real-Time Immuno-PCR (IPCR)
• Chromatin Immunoprecipitation (ChIP)
• Viral quantitation
• Pathogen detection including CMV detection rapid diagnosis of meningococcal
infection, penicillin susceptibility of Streptococcus pneumoniae, Mycobacterium
tuberculosis and its resistant strains and waterborne microbial pathogens in the
environment
• Radiation exposure assessment
• In vivo imaging of cellular processes
• DNA damage (microsatellite instability) estimation
• DNA damage (nuclear DNA) and DNA adduct estimation:
• Mitochondrial DNA studies (CNV, damage, deletion
• Methylation detection
• Measurement of unmethylated repeat DNA sequences
•
Detection of inactivation at X-chromosome• Determination of identity at highly polymorphic HLA loci
• Monitoring post transplant solid organ graft outcome
• Monitoring chimerism after hematopoietic stem cell transplantation
• Monitoring minimal residual disease after hematopoietic stem cell transplantation
• Determination of gene dosage and zygosity
• Genotyping by fluorescence melting-curve analysis (FMCA) or high-resolution melting
(HRM) analysis or specific probes/beacons reviewed in LNA or MGB probes can be
used allelic discrimination too
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References:
Lehmann, K. E., Buschmann, I. R., Unger, T., and Funke-Kaiser, H. (2006).
"Quantitative real-time RT-PCR data analysis: current concepts and the novel
"gene expression's CT difference" formula". J Mol Med 84: 901–10.
Boggy, G., and Woolf, P. J. (2010). Ravasi, Timothy. ed. "A Mechanistic Model
of PCR for Accurate Quantification of Quantitative PCR Data". PLOS One, 5 (8):
12355.
Sails AD (2009). "Applications in Clinical Microbiology". Real-Time PCR:
Current Technology and Applications. Caister Academic Press.
http://www.genomediagnostics.in/real-time-pcr.htm
http://pathmicro.med.sc.edu/pcr/realtime-home.htm
http://www.appliedbiosystems.com/absite/us/en/home/applications-
technologies/real-time-pcr/taqman-and-sybr-green-chemistries.html?ICID=EDI-
Lrn4
http://www.bio.davidson.edu/courses/genomics/method/realtimepcr.html