IN THE NAME OF GOD Alireza Azizi Alireza Salehi Mohsen Rahmanian

Real-Time PCR

Cockerill FR III. Arch Pathol Lab Med. 2003;127:1112

What is Wrong with Agarose Gels? * Poor precision * Low sensitivity * Short dynamic range < 2 logs * Low resolution * Non-automated * Size-based discrimination only * Results are not expressed as numbers * Ethidium bromide staining is not very quantitative

Real-Time PCR Definition:Real-time PCR is identical to a simple PCR except that the progress of the reaction in monitored by a camera or detector in “real time” Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production at each PCR cycle (in real time) as opposed to the endpoint detection Purpose: To estimate the initial quantity of specific template DNA.

Nigel Walker, NIEHS (www) (www) Log-view augments this part

Real-time PCR advantages * not influenced by non-specific amplification * amplification can be monitored real-time * no post-PCR processing of products * ultra-rapid cycling (30 minutes to 2 hours) * wider dynamic range of up to fold * confirmation of specific amplification by melting curve analysis * most specific, sensitive and reproducible * not much more expensive than conventional PCR (except equipment cost)

Real-time PCR disadvantages * setting up requires high technical skill and support * high equipment cost * * * * intra- and inter-assay variation * RNA lability * DNA contamination (in mRNA analysis)

Method of monitoring DNA amplification in real time Three general methods for the quantitative assays: 1. Hydrolysis probes (TaqMan, Beacons) 2. Hybridization probes (Light Cycler) 3. DNA-binding agents (SYBR Green)

Ampli taq DNA polymerase has 5`exo-nuclease activity. The 5`exo-nuclease activity of taq polymerase and FRET(Fluorescent Reasonant Energy Transfer) make it possible to detect PCR amplification in real-time. FRET: Fluorescent Reasonance Energy Transfer technology is utilized in the 5` nuclease assay. The principle is that when a high-energy dye is in close proximity to a low- energy dye there will be transfer of energy from high to low. TaqMan probes are oligonucleotides longer than the primers (20-30 bases long with a Tm value of 10 o C higher) that contain a high-energy dye termed a ‘Reporter’ at the 5' end, and a low-energy molecule termed a quenching dye (usually TAMRA or a non-fluorescent quencher (NFQ)) typically at the 3`. When this probe is intact and excited by light source the reporter dye’s emission is suppressed by the quncher dye as a result of close proximity of the dyes.(with FRET) Taq-man prob

When the probe is cleaved by the 5` nuclease activity of the enzyme the distance between Reporter and the Quncher increases causing the transfer of energy to stop. The fluorescent emission of the reporter increase and the Quencher decrease.

TaqMan Probes FRET = Förster/fluorescence resonance energy transfer & DNA Polymerase 5' exonuclease activity * Tm value 10 0 C higher than primers * runs of identical nucleotides (no consecutive Gs) * G+C content 30-80% * more Cs than Gs * no G at the 5' end

FRET = Förster/fluorescence resonance energy transfer

Mocellin et al. Trends Mol Med 2003 (www) (www) DNA Polymerase 5' Exonuclease Activity

The TaqMan 5’ Exonuclease Assay In addition to two conventional PCR primers, P1 and P2, which are specific for the target sequence, a third primer, P3, is designed to bind specifically to a site on the target sequence downstream of the P1 binding site. P3 is labelled with two fluorophores, a reporter dye (R) is attached at the 5 end, and a quencher dye (D), which has a different emission wavelength to the reporter dye, is attached at its 3 end. Because its 3’ end is blocked, primer P3 cannot by itself prime any new DNA synthesis. During the PCR reaction, Taq DNA polymerase synthesizes a new DNA strand primed by P1 and as the enzyme approaches P3, its 5’ exonuclease activity degrades the P3 primer from its 5’ end. The end result is that the nascent DNA strand extends beyond the P3 binding site and the reporter and quencher dyes are no longer bound to the same molecule. As the reporter dye moves away from the quencher, the resulting increase in reporter emission intensity is easily detected. Human Molecular Genetics 2. NCBI Books

Most common choices are FAM and VIC/HEX/JOE as reporters and TAMRA as quencher (or a NFQ)

Reporter, Quencher and Internal Reference Dyes * The classical reporter dye is 6-FAM (fluorescein) * Other reporters used for multiplexing are JOE and VIC * Some other real-time machines, such as the Stratagene Mx4000, can use red dyes as reporters * The classic quencher dye was TAMRA (rhodamine) but mainly replaced by non-fluorescent quenchers (NFQ) * Newer quenchers (NFQs, dark dyes) are DABYCL and the black hole quenchers (Biosearch Technologies) * TAMRA-quenched probes do not require a reference dye; they can use the TAMRA itself * Single probe reactions quenched by dark dyes (NFQ) should use an internal reference dye, classically ROX (dark red) * Multiplex reactions usually use dark quenchers and ROX

Real-Time PCR Principles Three general methods for the quantitative detection: 1. Hydrolysis probes (TaqMan, Beacons) 2. Hybridization probes (Light Cycler) 3. DNA-binding agents (SYBR Green)

Molecular beacon Molecular beacons also contain fluorescent (FAM, TAMRA, TET, ROX) and quenching dyes (typically DABCYL ) at either end but they are designed to adopt a hairpin structure while free in solution to bring the fluorescent dye and the quencher in close proximity for FRET to occur. They have two arms with complementary sequences that form a very stable hybrid or stem. The close proximity of the reporter and the quencher in this hairpin configuration suppresses reporter fluorescence. When the beacon hybridizes to the target during the annealing step, the reporter dye is separated from the quencher and the reporter fluoresces (FRET does not occur). Molecular beacons remain intact during PCR and must rebind to target every cycle for fluorescence emission. This will correlate to the amount of PCR product.

Molecular Beacons

Syber Green Sybr green is a dye which binds to double stranded DNA but not to single-stranded DNA and is frequently used to monitor the synthesis of DNA during real- time PCR reactions. When it is bound to double stranded DNA it fluoresces very brightly (much more brightly than ethidium bromide does.

SYBR Green (double-stranded DNA binding dye) * emits a strong fluorescent signal upon binding to double-stranded DNA * nonspecific binding is a disadvantage * requires melting point curve determination * longer amplicons create a stronger signal * may be multiplexed when coupled with melting curve analysis

SYBR Green (1) At the beginning of amplification, the reaction mixture contains the denatured DNA, the primers and the SYBR Green. The unbound dye molecules weakly fluoresce, producing a minimal background fluorescence signal which is subtracted during computer analysis. (2) After annealing of the primers, a few dye molecules can bind to the double strand. DNA binding results in a dramatic increase of the SYBR Green molecules to emit light upon excitation. (3) During elongation, more and more dye molecules bind to the newly synthesized DNA. If the reaction is monitored continuously, an increase in fluorescence is viewed in real-time. Upon denaturation of the DNA for the next heating cycle, the dye molecules are released and the fluorescence signal falls.

* Assays that do not require specificity of probe based assays. Detection of 1000s of molecules * When the PCR system is fully optimized -no primer dimers or non-specific amplicons, e.g. from genomic DNA When to Choose SYBR Green

When Not to Choose SYBR Green * Allelic discrimination assays (not an absolute one) * Multiplex reactions (not an absolute one) * Amplification of rare transcripts * Low level pathogen detection

Bustin SA. J Mol Endocrinol 2002 (www)(www)Scorpions

Bustin SA. J Mol Endocrinol 2002 (www)(www)Scorpions See also Didenko, Biotechniques 2001 (www)www

SCORPIONS Q R

Q R Q R Q R Q R Q R Q R Q R Q R Q R Q R R

The five-fold dilution series seems to plateau at the same place even though the exponential phase clearly shows a difference between the points along the dilution series. This reinforces the fact that if measurements were taken at the plateau phase, the data would not truly represent the initial amounts of starting target material. log view

Linear vs Log View log view linear view

Linear vs Log View linear viewlog view

Threshold Cycle * threshold cycle or the C T value is the cycle at which a significant increase in Rn is first detected * it is the parameter used for quantitation * C T value of 40 or more means no amplification and cannot be included in the calculations

The Amplification Plot contains valuable information for the quantitative measurement of DNA or RNA. The Threshold line is the level of detection or the point at which a reaction reaches a fluorescent intensity above background. The threshold line is set in the exponential phase of the amplification for the most accurate reading. The cycle at which the sample reaches this level is called the Cycle Threshold, C T. These two values are very important for data analysis using the 5’ nuclease assay. What is C T ? log view

Van der Velden. Leukemia 2003 (www) (www)

Rn * Rn + is the Rn value of a reaction containing all components (the sample of interest); Rn - is the Rn value detected in NTC (baseline value) * Rn is the difference between Rn + and Rn -. It is an indicator of the magnitude of the signal generated by the PCR * Rn is plotted against cycle numbers to produce the amplification curves and to estimate the C T values

(www) What is Rn?

Real-Time PCR Applications - I * quantitation of gene expression * array verification * quality control and assay validation * biosafety and genetic stability testing * drug therapy efficacy / drug monitoring * viral quantitation * pathogen detection

Real-Time PCR Applications - II * DNA damage (microsatellite instability) measurement * radiation exposure assessment * in vivo imaging of cellular processes * mitochondrial DNA studies * methylation detection * detection of inactivation at X-chromosome * linear-after-the-exponential (LATE)-PCR: a new method for real-time quantitative analysis of target numbers in small samples, which is adaptable to high throughput applications in clinical diagnostics, biodefense, forensics, and DNA sequencing

Real-Time PCR Applications - II * DNA damage (microsatellite instability) measurement * radiation exposure assessment * in vivo imaging of cellular processes * mitochondrial DNA studies * methylation detection * detection of inactivation at X-chromosome * linear-after-the-exponential (LATE)-PCR: a new method for real-time quantitative analysis of target numbers in small samples, which is adaptable to high throughput applications in clinical diagnostics, biodefense, forensics, and DNA sequencing

Real-Time PCR Applications - III * Determination of identity at highly polymorphic HLA loci * Monitoring post transplant solid organ graft outcome * Monitoring chimerism after HSCT * Monitoring minimal residual disease after HSCT * Genotyping (allelic discrimination) - Trisomies and copy number variations - Microdeletion genotypes - Haplotyping - Quantitative microsatellite analysis - Prenatal diagnosis from fetal cells in maternal blood - Intraoperative cancer diagnostics

Refrences Real time PCR, M.Tevfik Dorak Bachmann, M.H,Mathison-Dubard,genetic diversity of fline. Rapid Cycle Real-Time PCR-Methods and Applications (Wittwer Hahn, Kaul)Rapid Cycle Real-Time PCR-Methods and Applications Real-Time PCR: An Essential Guide (Edwards, Logan, Saunders)Real-Time PCR: An Essential Guide Real-Time PCR: Current Technology and Applications (Logan, Edwards, Saunders)Real-Time PCR: Current Technology and Applications Real-time PCR in Microbiology (MacKay IM)Real-time PCR in Microbiology