Real time PCR - European Pharmaceutical Review

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qPCR ISSUE 2009 Figure 8: Two sequence specific primers (top left corner), one for each allele of the polymorphic sequence to analyse, are used to generate PCR product, one being 10 (non-specific) base pair longer than the other. At then end of a PCR reaction, the two PCR product are therefore of different length as illustrated on a classic electrophoresis (top right corner). A melting curve assay relies on the slower release of SYBR-green from the longer PCR product. At the end of the reaction, PCR products are slowly heated. When double DNA start melting, SYBR-green is released and the fluorescence decrease accordingly (bottom left panel, fluorescence intensity). Speed of release is then calculated through a mathematical conversion (dF/dT), providing a melting curve where the pick is indicative of the melting temperature of the PCR product. Each sequence has a different melting point however; allele discrimination is brought by the additional 10 bp which provide an identifiable shift of melting point (bottom right corner). (Adapted from: Real-Time PCR. Chapter 8: Real-time PCR using SYBR® Green I by Dr Frederique Ponchel. Talyor &Francis. 2006 Oxford Press). In medical and biological research, the technique has also provided a lot of advances. Similar applications were used for similar and additional purposes such as assessing gene copy number in transgenic animals for examples 5 . One particular area that has considerably benefited from accurate quantification of DNA is chromatin immuno-precipitation (ChIP). ChIP is a powerful tool for the study of protein/DNA interactions. In this technique, specific protein/DNA complexes are immunoprecipitated using an antibody directed against the protein of interest. The proteins are then removed and the DNA is purified and analysed to determine which DNA fragments, but most importantly how much, was bound to the protein (see Figure 11 on page 14). Another area that has considerably benefited from real time PCR technology is the detection or identification of pathogens or contaminats. Toxins, virus (such as Hepatitis B presence or viral load 6 ) or bacteria (Chlamydia trachomatis presence and/or identification), genetically modified organisms can be detected/quantified either with real time PCR or end point Figure 9: Detection of gene amplification in DNA extracted from neuroblastoma tumor samples. We applied the SYBR-Green I assay to tumour samples for the detection of the MYC-N gene amplification. These samples had been tested previously by standard procedures for the diagnosis of neuroblastoma using fluorescence in-situ hybridisation (FISH)9. Real time PCR (black bars) compared well with the established amplification factor recorded for these tumours by FISH (open bars) and confirmed the presence of MYC-N amplification in six tumours. Adapted from BMC Biotechnology, 2003 5 12 www.europeanpharmaceuticalreview.com
SUBSCRIBE ISSUE 2009 qPCR assays. Tests were developed to detect mycotoxins, T-toxin, phytoestrogens such as Coumestrol and several other contaminants, particularly in food. Quantification of RNA This obviously is the area that has most immediately beneficiated from real time PCR. It is well accepted that measures of gene expression is now quantitative rather than semiquantitative. Two methods exist, one absolute comparing gene expression to known standards and a second, relative to an endogenous reference (usually a house keeping gene). Outside its plain use for gene expression studies (in cells in response to stimuli for example), real time PCR has been instrumental notably in transcriptome studies for confirming micro-array work, when limited amount of material is available (small tissue biopsy studies), or for large studies necessitation high thorough-put and automatism with PCR robots. This has notably warranted the development and success of the micro-fluidic card systems. It was recently argued 7 that advances in the tumour biology have now provided molecular targets for diagnosing and treating cancer. Clustering algorithms have revealed a molecular diversity that forms a new taxonomy with diagnostic, prognostic and possibly therapeutic significance. The challenge in pathology today is to develop and implement such molecular classifications in routine clinical practice. This requires objective, robust, and cost-effective techniques and real-time PCR offers such attractive features. Discrimination assay Genotyping using end point assays or melting curves analysis assays provides rapid results (raw biological samples to SNP genotyping results in less than one hour), highly accurate in virtually any sample (low amount of material or poor quality samples) and an increasing number of publications Figure 10: Pedigree of an Autosomal Dominant blindness family with a deletion of the entire OPA1 gene. Carriers of the deletion are indicated with filled symbols and normal individuals with open symbols 10 . Real-time was used in a copy number assay for the OPA1 gene using the SYBR-Green I dye. Quantification of the OPA1 gene deletion in 13 members of the family: Non-carriers showed a ratio close to 1 for the GAPDH versus OPA1 quantification. In contrast, all carriers of the deletion showed a clear reduction in this ratio. Adapted from BMC Biotechnology, 2003 5 (see for example any issues of Clinical Chemistry, Molecular Diagnostic, Nucleic Acid Research…) testifies how instrumental real time PCR has been in this field. The success of the technology has also promoted the development of new molecular tools such as minor grove binding, molecular beacon or scorpion probes. Genotyping assay for SNP are now available in array format, (TaqMan® SNP Genotyping Assays by ABI, Illumina Chip by Affimetrix and several others) making it straightforward to perform genome wide SNP genotyping studies. Importantly, melting curve analysis assays, scanning a particular sequence where polymorphisms are suspected, were shown to accurately reveal mutations/SNPs 8 . References 1. Luthra, R. and L.J. Medeiros, 5 '-> 3 ' exonuclease-based real-time PCR methods for detecting the t(14;18) and t(11;14) in non-Hodgkin's lymphomas. J. Clin. Ligand Assay, 2000; 23: 6-14. 2. Ramachandra, C. and S. Melnick, Multidrug resistance in human tumors: molecular diagnosis and clinical significance. Mol. Diag, 1999; 4: 81-94. 3. Vos, C.B.J., N.T. Ter Haar, J.L. Peterse, G.J. Cornelisse and M.J. Van de Vijver, Cyclin D-1 gene amplification and overexpression are present in ductal carcinoma in situ of the breast. J. Pathol., 1999; 187: 279-284. 4. Gunther, T., R. Schneider-Stock, C. Hackel, H.U. Kasper, M. Pross, GO TO CONTENTS PAGE 13
Page 1 and 2: TECHNOLOGY / KNOWLEDGE / INNOVATION
Page 3 and 4: EDITORIAL BOARD Dr Anthony Davies H
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