Novel Methods for High-Performance Melting Curve Analysis Using the LightCycler® 480 System

The LightCycler® Real-time PCR Systems provide fast, accurate, and versatile platforms for genetic variation research. They feature the temperature homogeneity and the optical characteristics re-quired for high-performance melting curve analysis. The plate-based LightCycler® 480 System now facilitates advanced applications in the emerging field of amplicon scanning for new mutations at high resolution.

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Melting-Curve-Based Analysis of Genetic Variation

Melting curve analysis is a well-established method for characterizing amplicons (e.g., for microbiological identification, or for detecting mutations and single nucleotide polymorphisms [SNPs]) [1]. The goal of SNP studies is either the detection of different allelic variants of a given SNP, or the identification of polymorphic positions at which previously unknown SNPs occur without determining the allele. Due to the modular design of the LightCycler® 480 System, both approaches can be pursued. A broad range of detection chemistries and data analysis algorithms add flexibility..

Homozygous and heterozygous samples can be differentiated easily using certain saturating DNA-binding dyes that do not interfere with amplification even at high concentrations and give very sharp melting profiles (Figure 1, 3) [2]. Once SNPs are known, their alleles are best analyzed by melting in the presence of sequence-specific labeled probes that bind with different affinities to different alleles, or allele combinations in the SNP-containing region (Figure 2).

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Melting curve raw data are generally represented by plotting fluorescence over temperature, with the data resolution and temperature range being higher with high resolution dyes than with labeled probes (Figure 1a, 2a). To make analysis more convenient, the negative first derivatives (-dF/dT) are often used, revealing melt-ing temperatures at peaks (Figure 1b, 2b). Data derived from homozygous wild types, homozygous mutants, and heterozygous samples can differ regarding peak number, peak position or a combination of both. With high-resolution dyes, differences between melting peaks are often detectable but not always large enough to enable clear differentiation of homozygotes (Figure 1b). Using sequence-specific probes allows for a more reliable separation (Figure 2b). The LightCycler® 480 Genotyping Software uses the latter approach for identifying SNP alleles.

Detection of Novel Sequence Variants

It is not possible to screen amplicons for unknown variations using sequence-specific probes, as the sequence neighboring the potential polymorphic site is unknown by definition. Novel generic DNA dyes can now be used instead and melting curve data acquired and analyzed at high resolution using LightCycler® 480 Gene Scanning Reagents and Software. In such an analysis, unknown sequence variations in diploids become apparent in heterozygous samples because they have significantly differently shaped melting curves compared to those derived from homozygous (wild type or mutant) samples. The LightCycler® 480 Instrument is the first plate-based real-time PCR instrument facilitating such analysis due to its high-performance thermal control, and optical components [3]. The novel LightCycler® 480 Gene Scanning Software analyzes these differences by first normalizing the data (see Figure 3a in comparison to Figure 1a) and then temperature-shifting the curves (Figure 3b). By plotting the difference in fluorescence between each sample and its respective wild type, heterozygote samples can be easily identified (Figure 3c). This method has been implemented in the novel LightCycler® 480 Gene Scanning Software.

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SNP Detection and Analysis Methods

Melting curve analysis is based on a robust, post PCR biophysical measurement and therefore has advantages over other mutation detection methods that derive information from the amplification process itself. Less sequence data is necessary in order to design a genotyping assay, more information can generally be revealed per reaction, and allele-specific primers or probes are not needed as the same dye or probe can be used for all alleles. Melting curve analysis with high-resolution dyes offers greater convenience and throughput than traditional methods (e.g., dHPLC) when amplicons are to be screened for the presence of novel genetic variants prior to or instead of sequencing.

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Conclusions

The LightCycler® 480 System is a versatile platform for the discovery and analysis of genetic variation. Due to its high-performance thermal control and optical components, it is currently the only plate-based real-time PCR system enabling mutation scanning by high-resolution melting curve analysis [3]. For both applications, genotyping and gene scanning, dedicated software analysis modules as well as ready-to-use, optimized master mixes have been developed as LightCycler® 480 solutions for genomic research.

References

1. von Ahsen N (2005) Clin Chem 51:1761–1762

2. Herrmann MG et al. (2006) Clin Chem 52:494–503

3. Dujos V et al. In: Tevfik D (ed) Real-Time PCR. Taylor and Francis, Abingdon (2006)

For additional information about the LightCycler® 480 System and the described mutation detection methods, please refer to: www.lightcycler480.com.

This article was originally published in Biochemica 1/2007, pages 17-19. ©Springer Medizin Verlag 2007