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LightCycler® 480 System - High Resolution Melting and Gene Scanning

 

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Introduction to High Resolution Melting Analysis

High Resolution Melting is a homogeneous, close-tube, post-PCR method, enabling researchers to analyze genetic variations (SNPs, mutations, methylations) in PCR amplicons. It goes beyond the power of classical melting curve analysis by studying the thermal denaturation of a double-stranded DNA in much more detail and with much higher information yield than ever before.

The most important High Resolution Melting application is gene scanning, the search for the presence of unknown variations in PCR amplicons prior to or as an alternative to sequencing. Mutations in PCR products are detectable by High Resolution Melting because they change the shape of DNA melting curves. A combination of new-generation DNA dyes, high-end instrumentation, and sophisticated analysis software permits the detections of these changes and the ability to derive information about the underlying sequence constellation.

By offering a High Resolution Melting Master PCR reagent and a special gene scanning analysis software for the LightCycler® 480 System, Roche provides a fully integrated, real-time PCR-based gene scanning solution in multiwell plates.

 

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Advantages of High Resolution Melting as a Method for Gene Scanning

High Resolution Melting provides high specificity and sensitivity and allows the processing of high sample numbers more conveniently and at much lower cost than traditional, non-homogeneous (gel-based) mutation screening methods (e.g., dHPLC) that require amplicons to be screened for variants on a separate instrument after PCR.

With High Resolution Melting, any amplicon can be screened for unknown sequence variants with a single high-resolution dye. Allele-specific primers or probes to target specific variants are not needed, and variants can be detected regardless of their position within the fragment.

Amplification reactions can be checked online for performance to make sure that analysis is based on samples with comparable DNA amount (similar Cp values).

 

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Why use the LightCycler® 480 System as a High Resolution Melting Platform?

The LightCycler® 480 System offers High Resolution Melting-based gene scanning as an integrated solution on a plate-based real-time PCR instrument. Hardware, software and dye-containing High Resolution Melting master mix have been developed in a concerted manner and are optimally designed to work together to support this novel application. The entire gene scanning experiment can be done on the same instrument for throughputs of up to 384 samples, and post-PCR analysis does not require a separate device. The power of HRM analysis on the LightCycler® 480 System has been well documented in numerous scientific publications.

To offer an integrated High Resolution Melting solution, Roche provides the following products:

 

The LightCycler® 480 Gene Scanning Software

As an additional analysis module for use in combination with the LightCycler® 480 Software 1.5:

  • Easy to use and integrates seamlessly into the LightCycler® 480 Software package.
  • Supports the use of melting standards for known genotypes to make analysis more reliable.
  • Allows easy access to other LightCycler® 480 analysis functions (e.g., Tm analysis to check PCR specificity, Cp calling to check PCR efficiency).

 

The LightCycler® 480 High Resolution Master

Optimized 2x concentrated hot start PCR master mix:

  • Contains a novel dye that is not toxic to amplification enzymes and can be used at only 0.8 nM working concentration.
  • Stable and robust in handling, for example, when used in automated workflows.
  • Minimal influence by temperature or irradiation in its fluorescence behavior.
  • Compatible with additives (e.g., DMSO) that enhance amplification of GC-rich sequences.
  • Separate 25 mM MgCl2 stock solution supplied with the Master for easy optimization of the Mg2+ concentration, if required.

 

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Application Examples

All experiments shown below were performed on the LightCycler® 480 Instrument using the LightCycler® 480 High Resolution Melting Master and analyzed using the LightCycler® 480 Gene Scanning module.

In general, human genomic DNA was purified from independent blood donors and PCR was run in triplicates. Analysis was done by normalization and temperature-shifting of fluorescence data, followed by plotting of the difference in fluorescence between each sample and a sample known to be wild type.

 

Amplicon Melting to Screen for Heterozygotes

Figure 1: High resolution melting curve analysis reveals differences between homozygous and heterozygous samples. A 163 bp fragment of the human ADD1 gene containing a G → T mutation was amplified from 96 independent blood samples in the presence of High Resolution Melting dye. Difference plot analysis allowed to differentiate between homozygous (wild type or mutant, blue) and heterozygous (red) samples.

 

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Homozygote Differentiation by Amplicon Melting

Figure 2: High resolution melting curve analysis reveals differences between wild type and variant sequences. A 163 bp fragment of the human LPLH3 gene containing a G → T mutation was amplified from 96 independent blood samples in the presence of High Resolution Melting dye. Difference plot analysis allowed to differentiate between wild type (green), homozygous mutant (red), and heterozygous (blue) samples. In cases where different homozygous forms are not always as readily distinguished as in this example (cf. Fig. 2), strategies are available to increase separation into distinct groups.

 

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High Resolution Melting Analysis Resolves Complex Patterns of Genetic Variation

Figure 3: High Resolution Melting analysis done on a 219 bp human MBL-2 gene fragment (384 samples) revealed a total of four main genetic subgroups (blue, red, green, and olive) as well as a few samples of two additional genotypes (orange and magenta).

 

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Probe-Free Diplotyping of two Adjacent SNPs

Figure 4: Segregation of haplotypes (combinations of adjacent SNPs) by High Resolution Melting. A 136 bp fragment of the human TNF alpha gene containing SNP-1: rs10489266 (A to G) and SNP-2: rs1234315 (C to T) was investigated. Analysis with the LightCycler® 480 Gene Scanning Software allowed to differentiate samples homozygous at both loci (green, red), heterozygous at only one (blue, cyan), or at both (pink) loci.

 

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Diplotyping of Adjacent SNPs Using an Unlabeled Probe

Figure 5: Diplotyping of adjacent SNPs using an unlabeled probe.

 

The same samples as in Figure 5 were melted in the presence of the depicted unlabeled 29 bp oligonucleotide. Analysis was done using the LightCycler® 480 Tm calling module to study the pattern of resulting melting peaks. Note that the analysis was done at much lower temperature than the amplicon melting in Figure 5. Allowing to make further distinctions within the groups of homo- or heterozygotes, respectively, the result obtained in presence of the probe goes beyond the one obtained with amplicon melting alone.

More recently, the power of HRM analysis for epigenetic studies has also been demonstrated. Roche´s NimbleGen arrays and the LightCycler® 480 System could be efficiently combined to screen genes involved in the control of  tumor formation and progression for unusual methylation patterns. For more information about this study, see Related Documents below.

 

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References

A list of published articles using the LightCycler® 480 System for HRM studies is available here.

 

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Related Downloads

Rapid High-Throughput Methylation Analysis Using the LightCycler® 480 System
We assessed the performance of the instrument combined with the LightCycler® 480 High Resolution Melting Master mix for DNA methylation analysis using the methylation-sensitive high resolution melting (MS-HRM) methodology.
Article (PDF, 172 KB)