Disadvantages of Traditional PCR 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 for staining is not very quantitative Post-PCR processing Dr. Salwa Hassan Teama
Real Time PCR Real-time PCR: DNA amplification analysis is monitored simultaneously over the course of thermocycling, the amplification product is detected as it accumulate. Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production during each PCR cycle (i.e., in real time). Dr. Salwa Hassan Teama
Principles of Real-Time Quantitative PCR Techniques Dr. Salwa Hassan Teama
Hybridization Probes Technique One probe is labelled with a donor fluorochrome at the 3 end and a second adjacent- probe is labelled with an acceptor fluorochrome. When the two fluorochromes are in close vicinity (1–5 nucleotides apart), the emitted light of the donor fluorochrome will excite the acceptor fluorochrome (FRET). This results in the emission of fluorescence, which subsequently can be detected during the annealing phase and first part of the extension phase of the PCR reaction. After each subsequent PCR cycle more hybridization probes can anneal, resulting in higher fluorescence signals.
Scropion Primer/Probes Scropion primers contain an attached fluorescence labelled tail that hybridize to an amplified target. This self-probing amplicon provides substantial benefits for rapid assays with short equilibration times. Dr. Salwa Hassan Teama
FRET (Fluorescence Resonance Energy Transfer) The principle is that when a high-energy dye is in close proximity to a low-energy dye, there will be a transfer of energy from high to low. Dr. Salwa Hassan Teama
Absolute Quantitation Require known concentrations of DNA standard molecules whose concentration is known absolutely, e.g. recombinant plasmid DNA (recDNA), genomic DNA, RT-PCR product, commercially synthesized big oligonucleotide. Absolute quantification determines the input copy number of the transcript of interest using standard curve.
Relative Quantitation/ Endogenous Reference Normalization to active reference (endogenous control); divide target amount by active reference amount. Normalize for difference in amount of total RNA added to a reaction. Compensate for different levels of PCR inhibition. Can be run in the same or in separate tubes.
Relative Quantitation/ Comparative Ct methodIf the efficiency of both reactions is the same nostandard curve required: Average for Ct target = target Ct Average for Ct end. Control= control Ct Target Ct - control Ct= Ct Ct (sample)- Ct (calibrator) = Ct Normalized target relative to calibrator
Nomenclature commonly used in real time quantitative RT-PCR: Baseline is defined as PCR cycles in which a reporter fluorescent signal is accumulating but is beneath the limits of detection of the instrument. ΔRn is an increment of fluorescent signal at each time point. The ΔRn values are plotted versus the cycle number. Threshold is an arbitrary level of fluorescence chosen on the basis of the baseline variability. A signal that is detected above the threshold is considered a real signal that can be used to define the threshold cycle (Ct) for a sample. Threshold can be adjusted for each experiment so that it is in the region of exponential amplification across all plots. Ct is defined as the fractional PCR cycle number at which the reporter fluorescence is greater than the threshold. The Ct is a basic principle of real time PCR and is an essential component in producing accurate and reproducible data.
How Real-Time PCR Quantitation Assays Work In the initial cycles of PCR, there is little change in fluorescence signal (The baseline). An increase in fluorescence above the baseline indicates the detection of accumulated target (The amplification plot). A fixed fluorescence threshold can be set above the baseline. The parameter Ct (threshold cycle) is defined as the fractional cycle number at which the fluorescence passes the fixed threshold. Dr. Salwa Hassan Teama
Clinical Oncology Detection and quantification of chromosomal translocation by real time PCR has been applied to monitor minimal residual disease or to show graft versus lymphoma effect. Predictive genetic testing and identification of relevant single nucleotide polymorphism (SNPs). Determination of identity at highly polymorphic HLA loci. Monitoring post transplant solid organ graft outcome. Monitoring chimerism after hematopoietic stem cell transplantation. Dr. Salwa Hassan Teama
Gene Therapy Gene transfer estimation Biodistribution of vectors Gene Expression Cytokines, receptors,…… Cancer Identification of micro- metastases or minimal residual disease in colorectal cancer, neuroblastoma, prostate cancer. Dr. Salwa Hassan Teama
Others Drug therapy efficacy / drug monitoring DNA damage (microsatellite instability) measurement In vivo imaging of cellular processes Mitochondrial DNA studies Methylation detection Detection of inactivation at X-chromosome Microdeletion genotypes Quantitative microsatellite analysis Prenatal diagnosis / sex determination using single cell isolated from maternal blood maternal circulation Prenatal diagnosis of hemoglobinopathies 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 Dr. Salwa Hassan Teama
Wide dynamic range of quantification(7-8 log decades)Advantage High technical sensitivity (<5 copies) High precision (<2% SD) No post PCR steps, thus minimized risk of cross contamination High throughput Multiplex approach possible. PCR product increases exponentiallyLimitation Variation increases with cycle number Increased variation after transformation to linear values Overlap of emission spectra Maximal four simultaneous reaction Increased risk of false negative results Dr. Salwa Hassan Teama
Real Time PCRThe development of real time PCR haseliminated the variability traditionallyassociated with quantitative PCR, thusallowing the routine and reliable quantificationof PCR product. Dr. Salwa Hassan Teama
References and Further Reading C A Heid, J Stevens, K J Livak, et al.Genome Res. Real Time Quantitative PCR. 1996 6: 986-994 Mhlanga MM, Malmberg L.Using molecular beacons to detect single-nucleotide polymorphisms with real-time PCR. Methods. 2001 Dec;25(4):463-71. Real time quantitative PCR.Holland, PM., Abramson,RD.,. et al.,1992. Clinical Chemistry.38, 462- 463. Dieter 2002. Quantification using Real Time PCR Technology; Application and limitation.Trends in molecular medicne. Vol.8 (6).June 2002. Abravaya K, Huff J, Marshall R, Merchant B, Mullen C, Schneider G, Robinson J. Molecular beacons as diagnostic tools: technology and applications. Clin Chem Lab Med. 2003 Apr;41(4):468-74. Bustin SA., Journal of Molecular Endcrinology (2002)29,23-39. www.endocrinology.org. Timothy Hughes, Michael Deininger ,… et al., Blood, 1 July. Vol. 108, Number 1.www.bloodjourna.org Real Time PCR. www.appliedbiosystem.org Real Time PCR. http://www.gene-quantification.de/strategy Real Time PCR. Trends in Molecular Medicine vol. 8 No.6 June 2002 http://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/real-time-pcr.html Dr. Salwa Hassan Teama
Image Citation S. Jähnichen. Molecular Beacons. 26 September 2005. http://en.wikipedia.org/wiki/File:Molecular_Beacons.jpg S. Jähnichen. Scorpions Probes. 28 September 2005 http://de.wikipedia.org/w/index.php?title=Datei:Scorpions_Probes.jpg&filetimestamp= 20050928181852 Real-Time qRT-PCR: Model of Real Time Quantitation Plot. http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechQPCR.shtml Quantification strategies in real-time RT-PCR. http://www.gene- quantification.de/strategy.html Dr. Salwa Hassan Teama