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The LightCycler® 480 real-time PCR system: a versatile

platform for genetic variation research
© 2008 Nature Publishing Group http://www.nature.com/naturemethods

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
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
methodological approaches.

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

otic cells requires fast, accurate and cost-effective methods for screen-
High-resolution
ing large numbers of samples or loci in parallel. Variations identified by Specific probes
melting dye
genomic sequencing or array studies need to be subsequently confirmed
and validated. Basic method: Advanced method: Whole-amplicon melting
endpoint melting curve (mutation scanning,
Real-time PCR has become a well established technology for this pur- genotyping genotyping methylation analysis

pose. The plate-based LightCycler 480 system offers a broad selection of

methods and applications (Fig. 1). b
The basic method (endpoint genotyping) is highly suitable for simple
experimental setups when well characterized single-nucleotide poly-
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 probesone for each allelein 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. (michael.hoffmann@roche.com). 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
Relative signal (%)

Relative signal (%)

100,000 100,000 100,000
80,000 80,000 90,000

Relative signal (%)

60,000 80,000 Methylated
60,000
70,000 100%
40,000 40,000 10%
60,000
20,000 20,000 50,000 1%
0 40,000 0.1%
0
76 77 78 79 80 84 85 86 87 88 30,000 0%

Temperature (ºC) 20,000

Temperature (ºC)
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)
© 2008 Nature Publishing Group http://www.nature.com/naturemethods

21,015 22,656
Relative signal difference

19,015 20,656
AT

Relative signal difference

17,015 18,656
366 T/C
Normalized and temperature-shifted difference plot
15,015 16,656
+563 C/T

Relative Signal Difference

13,015 14,656 3,862
11,015 12,656 366 T/C –6,138
10,656 –16,138
9,015 TT 563 C/T –26,138
7,015 8,656 –36,138
6,656 507 G/A –46,138
5,015
4,656 Wild type –56,138
3,015 –66,138
1,015 2,656
–76,138
–0,656 –86,138
–0,985 AA
–96,138
76 77 78 79 80 84 85 86 87 88 74 75 76 77 78 79 80 81 82 83 84 85
Temperature (ºC) Temperature (ºC) Temperature (ºC)

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
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
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
unmethylated cytosines. A fragment of the MGMT tumor suppressor gene was amplified from these treated DNA samples and analyzed by high-resolution melting. Melting
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).

Scanning Software and LightCycler 480 High Resolution Melting Conclusions and outlook
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.
binding characteristics allow it to be used at high concentrations Owing to its superior temperature homogeneity, flexible detection
without inhibiting PCR. Its homogeneous staining of target sequenc- channels and powerful software analysis modules, the LightCycler
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
1. Grievink, H. & Stowell, K.M. Identification of ryanodine receptor 1 single-
and enabled a broad range of applications in basic and clinical
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.
2. Mercier, C. et al. Toxic death case in a patient undergoing gemcitabine-
R. et al. Transferring PCRs to HRM assays on the LightCycler® 480
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. Biochemica and has not been peer reviewed. Nature Methods takes no responsibility for
1, 11–13; 2008). the accuracy or otherwise of the information provided.

ii | MARCH 2008 | NATURE METHODS