Methylation Analysis with the LightCycler® 480 Real-Time PCR System
Featured study: Analysis of promoter methylation in DNA repair genes
Michael Krypuy1, Tomasz K. Wojdacz2, and Alexander Dobrovic1,3
1Molecular Pathology Research and Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, Australia
2Institute of Human Genetics, University of Aarhus, Aarhus, Denmark
3Department of Pathology, University of Melbourne, Parkville, Australia
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DNA methylation is the addition of a methyl group to the 5-carbon position of cytosine preceding a guanine (a CpG dinucleotide), which results in 5-methyl cytosine. Regions with relatively high CpG dinucleotide density are distributed non-randomly in the human genome, with a preference for the promoter region of genes, and are called CpG islands . In cancer, there is alteration in the distribution of methylation in the genome . Regions that are normally methylated become unmethylated and regions that are normally unmethylated, such as promoter-associated CpG islands, become hypermethylated. This leads to a change of chromatin structure at the affected locus, resulting in gene silencing. Genes important for tumor progression are recurrently silenced by promoter hypermethylation, such as those involved in DNA repair, cell cycle control, and apoptosis.
Methylation-sensitive high-resolution melting (MS-HRM) is a technique that has been shown to be suitable for locus-specific assessment of DNA methylation . It involves PCR amplification of the region of interest from bisulfite-modified DNA using primers that amplify both methylated and unmethylated sequences. Bisulfite modification of DNA converts unmethylated cytosine to uracil but leaves 5-methyl cytosine unchanged, allowing determination of the methylation status of the region of interest following PCR amplification. In MS-HRM, PCR reactions are performed in the presence of a saturating DNA intercalating dye, and high-resolution melting (HRM) analyses take place after amplification to determine whether the amplicons originate from methylated or unmethylated variants of the template. MS-HRM allows assessment of methylation throughout the whole amplicon between the primers. As it is an closed-tube methodology, preliminary assessment of methylation can be performed very rapidly. For detailed information about methylation at specific CpG sites, bisulfitesequencing can be employed.
Changes in the DNA methylation status of certain genes in a tumor will be reflected by its biology. Therefore, rapid high-throughput methods to assess DNA methylation are of great value to researchers and clinicians . We examined the performance of MS-HRM assays for two DNA repair genes known to be affected by promoter methylation, FANCF and MGMT, on the LightCycler® 480 Instrument using the LightCycler® 480 High Resolution Melting Master mix.
Materials and Methods
Various cell line DNA samples were used as test samples. One µg of each DNA sample was bisulfite modified. Methylation standards were constructed by diluting 100% methylated control DNA (bisulfite modified) in a pool of normal DNA (bisulfite modified) at ratios of 50%, 25%, 10%, 5%,and 1%. MS-HRM Primers were designed as outlined by Wojdacz & Hansen . Reactions were performed in 96-well LightCycler® 480 plates using the LightCycler® 480 High Resolution Melting Master Mix.
Results and Discussion
Both MS-HRM assays were able to detect 1% methylated DNA in a background of unmethylated DNA. Figure 1 shows that the MGMT MS-HRM assay has clearer separation of methylation standards at the lower dilutions than the FANCF assay at the primer annealing temperature (+58°C) used in this experiment.
Figure 2 shows various cell lines screened for methylation using the FANCF and MGMT MS-HRM assays. Figure 2a shows that out of the nine cell lines screened for FANCF, only the ovarian cancer cell line 2008 showed methylation, as the melting profile for this sample resembles the 100% methylated control. All other cell lines studied were unmethylated for FANCF. Figure 2b shows that the breast cancer cell line MDA-MB435 is methylated for MGMT. The profile for this sample again closely resembles the 100% methylated control, suggesting that all CpG sites within the amplicon are methylated. The HS578T breast cancer cell line is interesting because its melt profile is distinct from both the unmethylated and methylated profiles. This profile is characteristic of heterogeneous methylation in the region examined.
Figure 1: Performance of FANCF and MGMT MS-HRM assays on the LightCycler® 480 Instrument.
(a) The FANCF MS-HRM assay with 100% methylated and unmethylated controls and methylation standards at 50%, 25%, 10%, 5%, and 1%. Data analyzed using both "Tm calling" and "Gene Scanning" software modules.
(b) The MGMT MS-HRM assay with 100% methylated and unmethylated controls and methylation standards at 50%, 25%, 10%, 5%, and 1%. Data analyzed using both "Tm calling" and "Gene Scanning" software modules.
Figure 2: Various cell lines screened for methylation using the FANCF and MGMT MS-HRM assays.
(a) FANCF MS-HRM assay showing the presence of DNA methylation in ovarian cancer cellline 2008.
(b) MGMT MS-HRM assay showing the presence of methylation in breast cancer cell lines MDA-MB435 and HS578T.
The LightCycler® 480 Instrument in combination with the LightCycler® 480 High Resolution Melting Master mix is a robust platform for performing methylation-sensitive high resolution melting (MS-HRM) experiments, allowing rapid and high-throughput studies of DNA methylation.
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-  Wojdacz TK, Dobrovic A (2007) Nucleic Acids Res 35:e41.
-  Dobrovic A In: Coleman WB, Tsongalis GJ (eds) Molecular Diagnostics for the Clinical Laboratorian, pp149–160. Humana Press, Totowa, USA (2005).
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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.
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