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The Lightcycler® 480 Real-Time PCR System

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The Lightcycler® 480 Real-Time PCR System ADVERTISING FEATURE APPLICATION NOTES The LightCycler® 480 real-time PCR system: a versatile platform for genetic variation research methods Real-time PCR is a well established technique for studying genetic variation using various probe-based methods for genotyping as well as high-resolution analysis of whole amplicons melted in the presence of saturating DNA dyes. The latter, relatively new, method allows screening for unknown mutations or .com/nature e DNA modifications. The LightCycler® 480 real-time PCR system is a multiwell plate–based instrument that provides integrated applications for detecting and characterizing genetic variation using all these .natur w methodological approaches. http://ww From endpoint fluorescence to melting curve analysis Today’s research on somatic, genetic and epigenetic variation in eukary- a Detecting known variants Detecting any variation oup r otic cells requires fast, accurate and cost-effective methods for screen- G High-resolution Specific probes ing large numbers of samples or loci in parallel. Variations identified by melting dye genomic sequencing or array studies need to be subsequently confirmed lishing and validated. Basic method: Advanced method: Whole-amplicon melting b endpoint melting curve (mutation scanning, Real-time PCR has become a well established technology for this pur- genotyping genotyping methylation analysis Pu pose. The plate-based LightCycler 480 system offers a broad selection of methods and applications (Fig. 1). b Nature The basic method (endpoint genotyping) is highly suitable for simple 8 experimental setups when well characterized single-nucleotide poly- 200 © morphisms (SNPs) and regions need to be analyzed, especially when commercial primers and probes are available. Allele-specific signals can be generated and acquired upon the completion of PCR, using two differently labeled hydrolysis probesone for each allelein the same reaction. Primers and probes can be readily combined with LightCycler 480 reagents, and the results can be analyzed with the LightCycler 480 Endpoint Genotyping Software module. For more advanced setups, melting curves obtained with fluorescent hybridization probes provide additional information about the sequence under study. Known SNPs present in PCR amplicons may be investigated Figure 1 | Overview of PCR-based methods for genetic variation research on the LightCycler 480 system. (a) Available applications, formats and methods. with sequence-specific, labeled probes (known as HybProbe probes) that (b) LightCycler 480 instrument, with exchangeable 96- and 384-well blocks. bind with different strengths to different alleles or allele combinations in the SNP-containing region, depending on whether there are mismatches that keep the probes from fully matching the target sequence. Because gation of several SNPs in parallel in multiplex assays if a different color signal generation and analysis are done after amplification, there is no is chosen for each SNP. HybProbe probes can also be designed to cover influence on PCR efficiency (for example, when DNA amounts or purity several SNPs under one probe, thus allowing haplotype analysis. Melting are an issue), making the method more robust than endpoint analysis. curve analysis can also account for unexpected sequence changes pres- Only one fluorescence channel is acquired per locus, allowing investi- ent in the investigated region under the probe. Michael Hoffmann, Oliver Geulen & Christian Weilke High-resolution melting using ResoLight dye Roche Applied Science, Nonnenwald 2, 82377 Penzberg, Germany. Correspondence When a region being investigated is suspected of containing vari- should be addressed to M.H. ([email protected]). ants whose exact position is not known, the LightCycler 480 Gene NATURE METHODS | MARCH 2008 | i ADVERTISING FEATURE APPLICATION NOTES a b c Normalized and temperature-shifted melting curves Normalized and temperature-shifted melting curves Normalized and temperature-shifted melting curves ) ) 100,000 100,000 100,000 80,000 80,000 90,000 ) Methylated 60,000 60,000 80,000 70,000 100% e signal (% 40,000 40,000 e signal (% 60,000 10% 20,000 20,000 50,000 1% e signal (% 0.1% 0 0 40,000 Relativ 76 77 78 79 80 Relativ 84 85 86 87 88 30,000 0% Temperature (ºC) 20,000 Temperature (ºC) Relativ 10,000 0 74 75 76 77 78 79 80 81 82 83 84 85 Normalized and temperature-shifted difference plot Normalized and temperature-shifted difference plot Temperature (ºC) 21,015 22,656 19,015 20,656 A T 17,015 18,656 366 T/C Normalized and temperature-shifted difference plot erence 15,015 16,656 +563 C/T 13,015 erence 14,656 3,862 methods 11,015 12,656 366 T/C –6,138 –16,138 10,656 erence 9,015 T T 563 C/T –26,138 7,015 8,656 –36,138 e signal diff 507 G/A 5,015 6,656 –46,138 e signal diff 4,656 Wild type –56,138 3,015 –66,138 1,015 2,656 Relativ –76,138 –0,656 e Signal Diff –0,985 –86,138 A A Relativ –96,138 76 77 78 79 80 84 85 86 87 88 74 75 76 77 78 79 80 81 82 83 84 85 .com/nature Relativ e Temperature (ºC) Temperature (ºC) Temperature (ºC) .natur Figure 2 | Applications of high-resolution melting analysis on the LightCycler 480 System. (a) A fragment of the TNFSF18 gene with the A/T polymorphism rs723858 was w amplified from different samples of human genomic DNA using the LightCycler 480 High Resolution Melting Master and analyzed by high-resolution melting. The LightCycler 480 Gene Scanning Software detects the differences in the melting curves resulting from sequence variations of the PCR products and allocates the samples to groups of the same sequence. The three genotypes are clearly separated, even though A/T polymorphisms, especially the homozygous variants (green and red curves), are hard to distinguish using alternative methods with strand-separation analysis. (b) A section of exon 3 of the CYP2C9 gene was amplified from different samples of human genomic http://ww DNA. High-resolution melting analysis revealed groups of samples with different sequence variations. Sequencing of only one sample of each group allowed the sequence determination for all samples. (c) Mixtures with different ratios of genomic DNA from a healthy donor and fully methylated DNA were treated with bisulfite to modify oup r unmethylated cytosines. A fragment of the MGMT tumor suppressor gene was amplified from these treated DNA samples and analyzed by high-resolution melting. Melting G curve evaluation allows a rough quantification of the portion of methylated DNA; even samples with only 0.1% methylated DNA can be clearly distinguished from completely unmethylated DNA. Original data kindly provided by T. K. Wojdacz and A. Dobrovic. (Peter MacCallum Cancer Centre, Melbourne, Australia). lishing b Scanning Software and LightCycler 480 High Resolution Melting Conclusions and outlook Pu Master reagent offer additional benefits. The master mix contains The LightCycler 480 real-time PCR system is a versatile platform LightCycler 480 ResoLight Dye, a DNA-binding dye whose special for the study of gene expression regulation and genetic variation4,5. Nature binding characteristics allow it to be used at high concentrations Owing to its superior temperature homogeneity, flexible detection 8 without inhibiting PCR. Its homogeneous staining of target sequenc- channels and powerful software analysis modules, the LightCycler 200 © es results in sharp melting signals, allowing differentiation between 480 system fulfills the requirements of both basic and advanced homo- and heterozygous samples or often even between homozygous genotyping methods. wild-type and mutant samples (Fig. 2a,b). This approach is especially Further information about the LightCycler 480 System and the muta- useful as a screening technique before sequencing—for example, as a tion detection approaches described is available on the Roche Applied cost-effective alternative to denaturing high-performance liquid chro- Science special interest page (http://www.lightcycler480.com). matography. LightCycler and HRM are trademarks of Roche. Patent and license High-resolution melting curve analysis (HRM) on the LightCycler disclaimer information is available online (http://www.lightcycler.com). 480 system has greatly simplified discovery and detection of SNPs and enabled a broad range of applications in basic and clinical 1. Grievink, H. & Stowell, K.M. Identification of ryanodine receptor 1 single- nucleotide polymorphisms by high-resolution melting using the LightCycler research—for example, on genes involved in human tumors (Vossen, 480 System. Anal. Biochem., published online 21 November 2007. R. et al. Transferring PCRs to HRM assays on the LightCycler® 480 2. Mercier, C. et al. Toxic death case in a patient undergoing gemcitabine- based chemotherapy in relation with cytidine deaminase downregulation. System—examples for BRCA1. Biochemica 4, 10–11; 2007) and Pharmacogenet. Genomics 17, 841–844 (2007). malignant hyperthermia1 and for pharmacogenetic studies2 (Evrard, 3. Chateigner-Boutin, A.L. & Small, I. A rapid high-throughput method for the detection and quantification of RNA editing based on high-resolution A. et al. Mutation scanning of the cytidine deaminase gene by high- melting of amplicons. Nucleic Acids Res. 35, e114 (2007). resolution melting curve analysis using the LightCycler® 480 system. 4. Van Eyken, E. et al. A new, easy, and rapid high-throughput detection method for the common GJB2 (CX26), 35delG mutation. Genet. Test. 11, 231–234 Biochemica 3, 13–14; 2007). Moreover, HRM has allowed scientists to (2007). find new ways to shed light on basic biological mechanisms in which 5. Fazio, G., Palmi, C., Rolink, A., Biondi, A. & Cazzaniga, G. PAX5/TEL acts as a transcriptional repressor causing down-modulation of CD19, enhances variations at levels other than the DNA primary sequence level are migration to CXCL12, and confers survival advantage in pre-BI cells. Cancer involved. These include RNA editing3 and epigenetic changes such Res. 68, 181–189 (2008). as DNA methylation (Fig. 2) (Krypuy, M. et al. Rapid high-throughput ® This article was submitted to Nature Methods by a commercial organization methylation analysis using the LightCycler 480 system.
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