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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