Transfection of cells is one of the main techniques used to influence gene expression. Most primary cells and human skeletal myoblasts (SkMC) in particular are very difficult to transfect, whereas for cell lines such as C2C12, many suitable transfect more
New Application for the LightCycler® 480 System: Gene Scanning by High-Resolution Melting
Roche Diagnostics GmbH
High-resolution melting (HRM) is a novel, homogeneous, post-PCR method, enabling genomic researchers to analyze genetic variations (SNPs, mutations, methylations) in PCR amplicons. 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 influence the shape of DNA melting curves. A combination of new-generation DNA dyes, high-end instrumentation and sophisticated analysis software allows to detect these changes and to derive information about the underlying sequence constellation.
Introducing the LightCycler® 480 High Resolution Melting Master, and a special analysis software for the LightCycler® 480 System, Roche Applied Science offers the first fully integrated, real-time PCR-based gene scanning solution in multiwell plates.
Advantages of High-Resolution Melting as a Method for Gene Scanning
High-resolution melting provides high specificity and sensitivity. Furthermore, it allows 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.
The LightCycler® 480 Gene Scanning Workflow
In a LightCycler® 480 Gene Scanning experiment, sample DNA is first amplified via real-time PCR in the presence of a proprietary saturating DNA dye contained in the LightCycler® 480 High Resolution Melting Master. A melting curve is then produced using high data acquisition rates, and data are finally analyzed using the LightCycler® 480 Gene Scanning Software Module.
The LightCycler® 480 Gene Scanning Software has been developed specifically to provide the most accurate analysis of high-resolution melting curves. The analysis is done in three basic steps: signal normalization, temperature shifting and difference plotting .
Why use the LightCycler® 480 System as Gene Scanning Platform?
The LightCycler® 480 System is currently the only available platform offering high-resolution melting-based gene scanning as an integrated solution on a plate-based real-time PCR instrument, including intuitive software and an optimized master.
Hardware, software, and dye-containing high-resolution melting master mix have been developed together 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 in less than two hours, and post-PCR analysis does not require a separate device.
References and further information
1. Hoffmann M et al. (2007) Biochemica 1:17–19
2. Hoffmann M et al. (2007) Nature Methods Application Notes an17–an18 (www.nature.com/app_notes/nmeth/index)
3. Herrmann MG et al. (2006) Clin Chem 52:494–503
4. Dujols V et al. High-resolution melting analysis for scanning and genotyping., in Real Time PCR. Tevfik D, ed., Taylor and Francis, Abingdon, 2006
5. Reischl U (2006) Clin Chem 52:1985–1987
This article was originally published in Biochemica 2/2007, pages 10-11. ©Springer Medizin Verlag 2007
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