Real time PCR - European Pharmaceutical Review

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qPCR ISSUE 2009 Figure 2: In a real time PCR assay, a forward and a reverse primer are used to generate a PCR reaction. Two fluorescent dyes, a reporter die in 5’ (R) and a quencher die in 3’ (Q) are attached to the “Taqman” probe. When both dyes are attached to the probe, the fluorescent emission of the reporter dye is quenched (top panel). During each extension cycle, the Taq DNA polymerase cleaves the probe in small fragments in order to proceed to primer extension. This frees the reporter dye from the template DNA and the proximity of the rest of the probe (middle panel). Once separated from the quencher, the reporter emits its characteristic fluorescence throughout the DNA polymerisation (bottom panel). This fluorescence is measured at each cycle of the PCR (Adapted from: ABI web site) (see later allelic discrimination). Two main approaches have been developed to detect fluorescence directly proportionally to the amount of the PCR product. The most popular, the TaqMAn assay or 5’-nuclease assay, is based on the specific hybridisation and degradation of a dually-labeled probe to the PCR product itself (see Figure 2). It relies on a 5’-exonuclease activity of the Taq polymerase, which cleaves the probe freeing a fluorescent dye that can be measured at each cycle of the assay. Several fluorescent dyes are available, Vic, Tet, Fam etc. Another approach uses the SYBR-Green I dye, which emits fluorescence only when bound to double stranded DNA (i.e. PCR products) (see Figure 3). Although both assays are potentially rapid and sensitive, their principles of detection and optimisation are different, as is the resulting price per assay. Independently of the chemistry used, read-outs are given as the number of PCR cycles necessary to achieve a given level of fluorescence or “cycle threshold” (Ct). Fluorescence is recorded at each PCR technique. A classic PCR reaction is limited by a “plateau” effect terminating prematurely the reaction and usually due to the exhaustion of reagents in the mix (primers, dNTP). When the target is abundant this plateau limits the quantification capability of the assay. In contrast when the template has a very low initial copy number, its abundance at the end of 40 cycles may still not be sufficient to be detectable using classic electrophoresis gels. Classic PCR is therefore efficient in allowing positive/negative detection but its range is limited when quantification is involved. The use of fluorescence allows the analysis of the amount of PCR products at each cycle of the reaction (i.e. real time PCR) as opposed to at the reaction end however, this can also be done for particular application Figure 3: SYBR-green molecules binding double stranded DNA (courtesy of ABI) and PCR reaction PCR product in the presence of SYBR-Green. At each cycle of the PCR, the SYBRgreen die binds only to the double stranded DNA only and emits fluorescence directly in relation with the amount of DNA. (Adapted from: Real-Time PCR. Chapter 8: Real-time PCR using SYBR® Green I. By Dr Frederique Ponchel. Talyor &Francis. 2006 Oxford Press) 08 www.europeanpharmaceuticalreview.com
SUBSCRIBE ISSUE 2009 qPCR Figure 4: Total fluorescence and transformation. At each cycles of the PCR, total florescence is measured across a large range of wavelength (top left corner, individual traces for fluorescence measures at four different cycles) at the end of the annealing / elongation phase (59oC) from cycle 5 till cycle 40 (bottom left corner). Individual traces indicate the three main components in this example. The pick to the right represents the buffer dye, ROX, which remain stable with time. The pick to the left increase with time and represents the amount of fluorescence release specifically at each cycle when quencher dye (TAMRA) is cleaved away from the reporter dye (FAM in this example). Total fluorescence traces are then transformed into individual fluorescence components (top right corner) where specific fluorescence are followed with time trough the progression of the PCR. Here, ROX fluorescence is stable (yellow line) indicating a reaction with no mis-happening. The TAMRA fluorescence (green line) represents the quencher and is also stable across the reaction. FAM (red line) increases with time, representing the PCR product. Finally, the multi-components analysis is transformed into an amplification plot (bottom right corner) as described in more details in Figure 5. phase of the PCR (linear part of the Log-fluorescence curve). As a Ct is proportional to the logarithm of the initial amount of target in a sample, the relative concentration of one target with respect to another is reflected in the difference in cycle number (ΔCt = Ct sample – Ct reference ) necessary to achieve the same level of fluorescence. A glossary of real time PCR terminology is available on the web. The main benefit of real time PCR which made its popularity (in addition to the fact that the assay last about two hours) is that its sensitivity usually allows detection over a 6 to 7 log scale (see Figure 6 on page 10). Furthermore, two reactions can be multiplexed in the same tube allowing a house-keeping gene to be measured under the exact same conditions as the test gene. In addition to real-time PCR as such, which allows quantification of PCR products at each cycle of the reaction, an “end point assay” ((see Figure 7 on page 10) most often referred to as allelic discrimination cycle across the whole wavelength range (see Figure 4). The algorithm within the real time PCR software then calculates the specific contribution to the total fluorescence of the reporting die, the quencher and the passive reference (see Figure 4). This is then transformed in an amplification plot where fluorescence is represented at each cycle of the PCR (see Figure 5). During the initial cycles, the fluorescence signal emitted by the die (either by the reporter of a TaqMan probe or by SYBR-green I) is usually too weak to register above background. During the exponential phase of the PCR, the fluorescence doubled at each cycle. A precise fluorescence doubling at each cycle is an important indicator of a well-optimised assay. After 30-35 cycles, the intensity of fluorescent signal usually began to plateau, indicating that the PCR had reached a saturation status. Cts are best recorded during the exponential Figure 5: PCR amplification plot under a log-phase scale. During the initial cycles (between 0 and ~15 cycles), the fluorescence signal emitted by the die is usually too weak to register above background. However, this is a very important phase of the assay as it determines how the algorithm calculating Cts at each cycle should consider as specific fluorescence above background to allocate values to the reporting die (FAM, VIC, SYBRgreen…) as opposed to the quencher (TAMRA in TaqMan assay) and the reference fluorescence (ROX). During the exponential phase of the PCR, the fluorescence precisely doubled at each cycle. After 30-35 cycles, the intensity of fluorescent signal began to plateau, indicating that the PCR had reached a saturation status. Cts are best recorded during the exponential phase of the PCR (linear part of the Log-fluorescence curve). (Adapted from: Real-Time PCR. Chapter 8 : Real-time PCR using SYBR® Green I. By Dr Frederique Ponchel. Talyor &Francis. 2006 Oxford Press) GO TO CONTENTS PAGE 09
Page 1 and 2: TECHNOLOGY / KNOWLEDGE / INNOVATION
Page 3 and 4: EDITORIAL BOARD Dr Anthony Davies H
Page 5 and 6: ISSUE ONE 2009 Contents Index to Ad
Page 7: © 2009 Thermo Fisher Scientific In
Page 11 and 12: SUBSCRIBE ISSUE 2009 qPCR chromosom
Page 13 and 14: SUBSCRIBE ISSUE 2009 qPCR assays. T
Page 15 and 16: Single Cell Gene Expression from Fl
Page 17 and 18: © 2008 Thermo Fisher Scientific In
Page 19 and 20: SUBSCRIBE ISSUE 2009 HIGH CONTENT S
Page 21 and 22: SUBSCRIBE ISSUE 2009 HIGH CONTENT S
Page 23 and 24: More Imaging Speed Introducing the
Page 25 and 26: SUBSCRIBE ISSUE 2009 LAB AUTOMATION
Page 27 and 28: ISSUE 2009 LAB AUTOMATION washed su
Page 29 and 30: SUBSCRIBE ISSUE 2009 THERMAL ANALYS
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Page 33 and 34: SUBSCRIBE ISSUE 2009 PAT a bioproce
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Page 37 and 38: SUBSCRIBE ISSUE 2009 PAT extensive
Page 39: PharmaINFOcus A REGULAR ROUND-UP OF

Real time PCR - European Pharmaceutical Review

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