PCR ( real-time polymerase chain reaction (Real-Time PCR) Basic Princiable & Opertation

History of Real Time PCR

1989 Chehad et al developed a method which allowed PCR products to be detected, using fluorescent primers. However, the primers had to be removed downstream (at the end of the reaction) to visualise the results.

1990 Kellogg and Pang independently measured PCR product quantity in the log phase of the reaction, which required that each sample amplified with identical efficiency

1991 An internal control inhibitor is added to each reaction, which normalises the efficiency of the reaction; so can compare the target sequence with the inhibitor sequence. Holland et al developed a technique which caused a labelled probe to be cleaved once the desired sequence was made. However, a downstream process was needed to detect the products.

1992 Higuchi et al developed a truly homogeneous (combining amplification and detection in one reaction tube) assay for PCR and PCR product detection. Fluorescence is produced when dye like ethidium bromide (EtBr) binds to dsDNA, so the addition of EtBr to a PCR reaction causes fluorescence to increase in proportion to dsDNA strands.Found it possible to use a bifurcated fibre optic device to detect fluorescence from EtBr, and transmit it to a spectrofluorometer, in order to quantify in real time.

1993 Higuchi et al improved on their previous design, using a video camera to monitor MULTIPLE PCRs simultaneously (fibre optic method could only monitor 1).

1996 Heid et al developed the Taqman reaction (the fluorogenic probe reaction), which involves a hybridisation probe labelled with 2 different dyes. This allows much greater specificity than intercalating dyes like EtBr. Heid then used the real-time PCR to transcribe, amplify, detect and quantify Cystic Fibrosis Transmembrane Regulator mRNA in real time

2001 Michael Pfaffl devised a way to quantify mRNA levels, without doing an internal dilution curve for both the standard and reference sequence each time an equation called the Pfaffl Method.

Present Fluoregenic probe reaction is still used today, with SYBR Green I dye instead of EtBr. The real-time monitoring of the fluorescence allows measurement of the amplification progress of the sample.

What are Fluorescent dyes?

When a population of fluorochrome molecules is excited by light of an appropriate wavelength, fluorescent light is emitted. The light intensity can be measured by flurometer or a pixel-by-pixel digital image of the sample. Excitation and Emission: Fluorodyes absorb light at one wavelength & thereby boosts an electron to a higher energy shell.

? The excited electron falls back to the ground state and the flurophore re- emits light but at longer wavelength.

? This shift makes it possible to separate excitation light from emission light with the use of optical filters.

? The wavelength (nm) where photon energy is most efficiently captured is defined as the Absorbancemax & the wavelength (nm) where light is most efficiently released is defined as the Emissionmax.

The range for which flurodyes absorb light is small (~ < 50nm) and light outside this range will not cause the molecule to fluoresce. Linearity: The intensity of the emitted fluorescent light is a linear function of the amount of fluorochrome present. The signal becomes nonlinear at very high fluorochrome concentrations. Brightness: Fluorochrome differ in intensity. Dull fluorochrome is a less sensitive probe than a bright fluorochrome. The brightness depends on two properties of the fluorochrome-

?Its ability to absorb light (extinction coefficient).

?The efficiency with which it converts absorbed light into emitted fluorescent light (quantum efficiency).

Environmental factors: Environmental conditions can affect the brightness or the wavelength of the absorption or emission peaks.

Fluorescence Resonance Energy Transfer

FRET is a distance dependent interaction between the excited states of 2 dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon

The Donor and Acceptor in close physical proximity (10 -100 Angstrom) can lead to FRET or Quenching

Quantitating Fluorescence

A flurometer exploits the principles of fluorescence to quantitate fluorescent (dye) molecules in the following way:

A strong light source which produces light within a specific light range ( eg xenon arc lamp) is focused down to a tight beam. The tight beam of light is sent through a filter which removes most of the light outside of the target wavelength range. The filtered light beam passes through the liquid target sample striking some of the fluorescent molecules in the sample. Light emitted from the fluorescent molecules travels orthogonal to the excitation light beam pass through a secondary filter that removes most of the light outside of the target wavelength range. The filtered light then strikes a photodetector or photomultiplier which allows the instrument to give a relative measurement of the intensity of the emitted light.

Components of PCR

The PCR reaction requires the following components:

1.DNA Template : The double stranded DNA (dsDNA) of interest, separated from the sample.

2. DNA Polymerase : Usually a thermostable Taq polymerase that does not rapidly denature at high temperatures (98°), and can function at a temperature optimum of about 70°C.

3. Oligonucleotide primers : Short pieces of single stranded DNA (often 20-30 base pairs) which are complementary to the 3’ ends of the sense and anti-sense strands of the target sequence.

4. Deoxynucleotide triphosphates : Single units of the bases A, T, G, and C (dATP, dTTP, dGTP, dCTP) provide the energy for polymerization and the building blocks for DNA synthesis.

5.Buffer system : Includes magnesium and potassium to provide the optimal conditions for DNA denaturation and renaturation; also important for polymerase activity, stability and fidelity.

Procedure of PCR

A. Amplification

1- Denaturation

High temperature incubation is used to “melt” double- stranded DNA into single strands and loosen secondary structure in single-stranded DNA. The highest temperature that the DNA polymerase can withstand is typically used (usually 95°C). The denaturation time can be increased if template GC content is high.

2- Annealing

During annealing, complementary sequences have an opportunity to hybridize, so an appropriate temperature is used that is based on the calculated melting temperature (Tm) of the primers(5°C below the Tm of the primer).

3-Extension

At 70-72°C, the activity of the DNA polymerase is optimal, and primer extension occurs at rates of up to 100 bases per second. When an amplicon in real-time PCR is small, this step is often combined with the annealing step using 60°C as the temperature

B. Detection

The detection is based on fluorescence technology. The specimen is first kept in proper well and subjected to thermal cycle like in the normal PCR. The machine, however, in the Real Time PCR is subjected to tungsten or halogen source that lead to fluoresce the marker added to the sample and the signal is amplified with the amplification of copy number of sample DNA. The emitted signal is detected by an detector and sent to computer after conversion into digital signal that is displayed on screen. The signal can be detected when it comes up the threshold level (lowest detection level of the detector)

Instrument for PCR

• LightCycler :-technology combines rapid-cycle polymerase chain reaction with real-time fluorescent monitoring and melting curve analysis. Since its introduction in 1997, it is now used in many areas of molecular pathology, including oncology (solid tumors and hematopathology), inherited disease, and infectious disease. By monitoring product accumulation during rapid amplification, quantitative polymerase chain reaction in a closed-tube system is possible in 15 to 30 minutes. Furthermore, melting curve analysis of probes and/or amplicons provides genotyping and even haplotyping. Novel mutations are identified by unexpected melting temperature or curve shape changes. Melting probe designs include adjacent hybridization probes, single labeled probes, unlabeled probes, and snapback primers. High-resolution melting allows mutation scanning by detecting all heterozygous changes. This review describes the major advances throughout the last 15 years regarding LightCycler technology and its application in clinical laboratories.

Monitoring PCR in real time using DNA dyes, hydrolysis probes, and hybridization probes. The top row shows data collected once each PCR cycle, and the bottom row shows data collected continuously (five times per second) during all PCR cycles.

• The Rotor-Gene : is unlike any other instrument. It was designed from the ground-up for real-time thermo-optical analysis. The key difference is the unique centrifugal rotary design that ensures well-to-well variation is negligible — as it should be. In the Rotor-Gene, every tube spins quickly in a chamber of moving air. Thus there is no positional temperature variation such as the recognized “edge effect” observed in block-based instruments. Optically, the Rotor-Gene is similarly uniform because every tube moves past the identical excitation and detection optics. 
• iCycler :-

The optical module houses the excitation system and the detection system. The Excitation system consists of a fan-cooled, 50-watt tungsten halogen lamp, a heat filter (infrared absorbing glass), a 6-position filter wheel fitted with optical filters and opaque filter “blanks”, and a dual mirror arrangement that allows simultaneous illumination of the entire sample plate. The excitation system is physically located on the right front corner of the optical module, with the lamp shining from right to left, perpendicular to the instrument axis. Light originates at the lamp, passes through the heat filter and a selected color filter, and is then reflected onto the 96 well plate in the thermal cycler by a set of mirrors. This light source excites the fluorescent molecules in the wells.

The detection system occupies the rear two-thirds of the optical module housing. The primary detection components include a 6-position emission filter wheel, an image intensifier, and a CCD detector. This filter wheel is identical to the wheel in the excitation system and is fitted with colored emission filters and opaque filter “blanks”. The intensifier increases the light intensity of the fluorescence without adding any electrical noise. The 350,000 pixel CCD allows very discrete quantitation of the fluorescence in the wells. Fluorescent light from the wells passes through the emission filter and intensifier and is then detected by the CCD.

• Multiplex Quantitative PCR System

Multiplex PCR is a widespread molecular biology technique for amplification of multiple targets in a single PCR experiment. In a multiplexing assay, more than one target sequence can be amplified by using multiple primer pairs in a reaction mixture. As an extension to the practical use of PCR, this technique has the potential to produce considerable savings in time and effort within the laboratory without compromising on the utility of the experiment.

Types of Multiplex PCR

Multiplexing reactions can be broadly divided in two categories:

1. Single Template PCR Reaction

This technique uses a single template which can be a genomic DNA along with several pairs of forward and reverse primers to amplify specific regions within a template.

2. Multiple Template PCR Reaction

It uses multiple templates and several primer sets in the same reaction tube. Presence of multiple primers may lead to cross hybridization with each other and the possibility of mis-priming with other templates. 

Data showing log amplification plots of a set of four 4-fold serial dilutions of hgDNA ranging from 40 – 0.625 ng per reaction using a fast 2-step cycling protocol. Data represents four replicates for each DNA dilution. The ActB and ERBB2 assays are shown in green and orange, respectively.

• Sequence Detection Systems Quantitative Assay :-

A comprehensive set of guidelines covering assay design and optimization has been developed by Biosystems to ensure success when using Sequence Detection Systems instrumentation. These guidelines, however, remain simple and easy to follow. Furthermore, many variables that have required optimization in traditional PCR are kept constant, reducing assay setup and development time. The assay design and optimization procedure contains the following important steps:

? Primer and probe design using Primer Express? software

? Selecting the appropriate reagent configuration

? Universal thermal cycling parameters

? Assay optimization

• SmartCycler

Some Microorganisms take longer to detect, with higher limits up to 104 cells/ml. A commercial version of this instrument, called the Smartcycler, is now available from Cepheid in Sunnyvale, CA, with 96 separate individually programmable multiplex reaction chambers. Although the Smartcycler is not specifically field portable, Cepheid has designed a notebook-sized (3.3 kg) real-time PCR field portable unit that they tested for detection of Bacillus subtilis and Bacillus thuringiensis DNA and that uses independently programmable real-time multiplexed PCR chambers . The ANAA, like the MATCI, requires the DNA sample to be prepared prior to adding it to a real-time PCR reaction mix that is inserted into the machine, and can only be used by personnel experienced in PCR methods

Example Resulat : Reproducibility of the mPCR methods.

Mixed CRV and CEV plasmids were diluted as templates from 1×105 to 1×103 copies/μl to amplify specific fragments by using three different PCR instruments at different times. (A) CRV: Lane M, 100 bp DNA ladder; Lanes 1, 5, and 9, negative controls for the three tests; Lanes 2~4, mPCR amplifying 1×105 copies/μl, 1×104 copies/μl, and 1×103 copies/μl CRV plasmids; Lanes 6~8, mPCR amplifying different dilutions of mixed plasmids a second time; Lanes 10~12, mPCR amplifying different dilutions of mixed plasmids a third time. (B) CEV: Lane M, 100 bp DNA ladder; Lanes 1, 5, and 9, negative controls for the three tests; Lanes 2~4, mPCR amplifying 1×105 copies/μl, 1×104 copies/μl, and 1×103 copies/μl CEV plasmids; Lanes 6~8, mPCR amplifying different dilutions of mixed plasmids a second time; Lanes 10~12, mPCR amplifying different dilutions of mixed plasmids .

Applications PCR

• Viral Quantitation
• Quantitation of Gene Expression
• Array Verification
• Pathogen detection Drug
• Therapy Efficacy
• Genotyping

Website references

European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF): https://gmo-crl.jrc.ec.europa.eu/.

International Service for the Acquisition of Agri-biotech Applications (ISAAA): https://www.isaaa.org/gmapprovaldatabase/.

EU Register of Authorised GMOs: https://ec.europa.eu/food/dyna/gm_register/index_en.cfm.

EU-RL GMFF database on GMO detection methods: https://gmocrl.jrc.ec.europa.eu/gmomethods/.

GMDD: a database of GMO detection methods (Dong et al., 2008): https://gmdd.shgmo.org/. IRMM

application note 4: https://irmm.jrc.ec.europa.eu/reference_materials_catalogue/user_support/erm_application_notes/ application_note_4/Documents/application_note_4_english.pdf.

References

https://www.mun.ca/biology/scarr/Principle_of_RT-PCR.html

https://www.primerdesign.co.uk/assets/files/beginners_guide_to_real_time_pcr.pdf

https://link.springer.com/chapter/10.1007%2F978-90-481-3132-7_3

https://www.slideshare.net/pratyayseth/real-time-pcr-34159486

https://www.thermofisher.com/np/en/home/life-science/pcr/real-time-pcr/real-time-pcr-learning-center/real-time-pcr-basics/essentials-real-time-pcr.html

https://www.sciencedirect.com/topics/neuroscience/real-time-polymerase-chain-reaction