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Rapid analysis of heterogeneously methylated DNA using digital methylation-sensitive high resolution melting: application to the CDKN2B (p15) gene.

Candiloro IL, Mikeska T, Hokland P, Dobrovic A - Epigenetics Chromatin (2008)

Bottom Line: It eliminates both PCR and cloning bias towards either methylated or unmethylated DNA.Potentially complex information is simplified into a digital output, allowing counting of methylated and unmethylated alleles and providing an overall picture of methylation at the given locus.Downstream sequencing is minimised as dMS-HRM acts as a screen to select only methylated clones for further analysis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Pathology Research and Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, Victoria 8006, Australia. ida.Candiloro@petermac.org

ABSTRACT

Background: Methylation-sensitive high resolution melting (MS-HRM) methodology is able to recognise heterogeneously methylated sequences by their characteristic melting profiles. To further analyse heterogeneously methylated sequences, we adopted a digital approach to MS-HRM (dMS-HRM) that involves the amplification of single templates after limiting dilution to quantify and to determine the degree of methylation. We used this approach to study methylation of the CDKN2B (p15) cell cycle progression inhibitor gene which is inactivated by DNA methylation in haematological malignancies of the myeloid lineage. Its promoter region usually shows heterogeneous methylation and is only rarely fully methylated. The methylation status of CDKN2B can be used as a biomarker of response to treatment. Therefore the accurate characterisation of its methylation is desirable.

Results: MS-HRM was used to assess CDKN2B methylation in acute myeloid leukaemia (AML) samples. All the AML samples that were methylated at the CDKN2B promoter (40/93) showed varying degrees of heterogeneous methylation. Six representative samples were selected for further study. dMS-HRM was used to simultaneously count the methylated alleles and assess the degree of methylation. Direct sequencing of selected dMS-HRM products was used to determine the exact DNA methylation pattern and confirmed the degree of methylation estimated by dMS-HRM.

Conclusion: dMS-HRM is a powerful technique for the analysis of methylation in CDKN2B and other heterogeneously methylated genes. It eliminates both PCR and cloning bias towards either methylated or unmethylated DNA. Potentially complex information is simplified into a digital output, allowing counting of methylated and unmethylated alleles and providing an overall picture of methylation at the given locus. Downstream sequencing is minimised as dMS-HRM acts as a screen to select only methylated clones for further analysis.

No MeSH data available.


Related in: MedlinePlus

Sequencing analysis of dMS-HRM products from an acute myeloid leukaemia sample. PCR clones from sample 9164 were selected for sequencing as it showed the most variation, and was therefore the best example of the relationship of peak position to the degree of methylation. One representative of each peak observed from dMS-HRM is shown (a). Peaks are grouped within colours according to the number of CpG dinucleotides methylated as shown by sequencing and represented by lollipops, where open and filled circles represent unmethylated and methylated CpG sites, respectively (b). The order of clones in panel (b) reflects the order of peaks from left to right. The asterisk (*) marks PCR clone 69, as although it showed complete methylation, it also contained an unconverted cytosine. This is shown in the sequencing trace in the centre panel of (c), compared with an unmethylated sequence (above) and a methylated sequence (below). The incompletely converted cytosine is indicated in red. This incomplete conversion explains the higher Tm of this clone with respect to the other clones showing complete methylation.
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Figure 4: Sequencing analysis of dMS-HRM products from an acute myeloid leukaemia sample. PCR clones from sample 9164 were selected for sequencing as it showed the most variation, and was therefore the best example of the relationship of peak position to the degree of methylation. One representative of each peak observed from dMS-HRM is shown (a). Peaks are grouped within colours according to the number of CpG dinucleotides methylated as shown by sequencing and represented by lollipops, where open and filled circles represent unmethylated and methylated CpG sites, respectively (b). The order of clones in panel (b) reflects the order of peaks from left to right. The asterisk (*) marks PCR clone 69, as although it showed complete methylation, it also contained an unconverted cytosine. This is shown in the sequencing trace in the centre panel of (c), compared with an unmethylated sequence (above) and a methylated sequence (below). The incompletely converted cytosine is indicated in red. This incomplete conversion explains the higher Tm of this clone with respect to the other clones showing complete methylation.

Mentions: Six AML samples that represented the range of variation observed were analysed digitally. Figure 3 shows the digital melting profiles of the selected AML samples. 4278 does not show any methylated alleles. 4383 and 4156 show products that correspond either to unmethylated products or have only few CpG sites methylated. 3224, 9164 and 730-06 cover a broad range of heterogeneously methylated alleles. Selected amplicons from one sample, 9164, were chosen for sequencing as it displayed the most variation in dMS-HRM products (Figure 4a). There is a close relationship between the Tm of the melting peaks and the number of CpG sites methylated, which vary only in the CpG sites being methylated.


Rapid analysis of heterogeneously methylated DNA using digital methylation-sensitive high resolution melting: application to the CDKN2B (p15) gene.

Candiloro IL, Mikeska T, Hokland P, Dobrovic A - Epigenetics Chromatin (2008)

Sequencing analysis of dMS-HRM products from an acute myeloid leukaemia sample. PCR clones from sample 9164 were selected for sequencing as it showed the most variation, and was therefore the best example of the relationship of peak position to the degree of methylation. One representative of each peak observed from dMS-HRM is shown (a). Peaks are grouped within colours according to the number of CpG dinucleotides methylated as shown by sequencing and represented by lollipops, where open and filled circles represent unmethylated and methylated CpG sites, respectively (b). The order of clones in panel (b) reflects the order of peaks from left to right. The asterisk (*) marks PCR clone 69, as although it showed complete methylation, it also contained an unconverted cytosine. This is shown in the sequencing trace in the centre panel of (c), compared with an unmethylated sequence (above) and a methylated sequence (below). The incompletely converted cytosine is indicated in red. This incomplete conversion explains the higher Tm of this clone with respect to the other clones showing complete methylation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2590600&req=5

Figure 4: Sequencing analysis of dMS-HRM products from an acute myeloid leukaemia sample. PCR clones from sample 9164 were selected for sequencing as it showed the most variation, and was therefore the best example of the relationship of peak position to the degree of methylation. One representative of each peak observed from dMS-HRM is shown (a). Peaks are grouped within colours according to the number of CpG dinucleotides methylated as shown by sequencing and represented by lollipops, where open and filled circles represent unmethylated and methylated CpG sites, respectively (b). The order of clones in panel (b) reflects the order of peaks from left to right. The asterisk (*) marks PCR clone 69, as although it showed complete methylation, it also contained an unconverted cytosine. This is shown in the sequencing trace in the centre panel of (c), compared with an unmethylated sequence (above) and a methylated sequence (below). The incompletely converted cytosine is indicated in red. This incomplete conversion explains the higher Tm of this clone with respect to the other clones showing complete methylation.
Mentions: Six AML samples that represented the range of variation observed were analysed digitally. Figure 3 shows the digital melting profiles of the selected AML samples. 4278 does not show any methylated alleles. 4383 and 4156 show products that correspond either to unmethylated products or have only few CpG sites methylated. 3224, 9164 and 730-06 cover a broad range of heterogeneously methylated alleles. Selected amplicons from one sample, 9164, were chosen for sequencing as it displayed the most variation in dMS-HRM products (Figure 4a). There is a close relationship between the Tm of the melting peaks and the number of CpG sites methylated, which vary only in the CpG sites being methylated.

Bottom Line: It eliminates both PCR and cloning bias towards either methylated or unmethylated DNA.Potentially complex information is simplified into a digital output, allowing counting of methylated and unmethylated alleles and providing an overall picture of methylation at the given locus.Downstream sequencing is minimised as dMS-HRM acts as a screen to select only methylated clones for further analysis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Pathology Research and Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, Victoria 8006, Australia. ida.Candiloro@petermac.org

ABSTRACT

Background: Methylation-sensitive high resolution melting (MS-HRM) methodology is able to recognise heterogeneously methylated sequences by their characteristic melting profiles. To further analyse heterogeneously methylated sequences, we adopted a digital approach to MS-HRM (dMS-HRM) that involves the amplification of single templates after limiting dilution to quantify and to determine the degree of methylation. We used this approach to study methylation of the CDKN2B (p15) cell cycle progression inhibitor gene which is inactivated by DNA methylation in haematological malignancies of the myeloid lineage. Its promoter region usually shows heterogeneous methylation and is only rarely fully methylated. The methylation status of CDKN2B can be used as a biomarker of response to treatment. Therefore the accurate characterisation of its methylation is desirable.

Results: MS-HRM was used to assess CDKN2B methylation in acute myeloid leukaemia (AML) samples. All the AML samples that were methylated at the CDKN2B promoter (40/93) showed varying degrees of heterogeneous methylation. Six representative samples were selected for further study. dMS-HRM was used to simultaneously count the methylated alleles and assess the degree of methylation. Direct sequencing of selected dMS-HRM products was used to determine the exact DNA methylation pattern and confirmed the degree of methylation estimated by dMS-HRM.

Conclusion: dMS-HRM is a powerful technique for the analysis of methylation in CDKN2B and other heterogeneously methylated genes. It eliminates both PCR and cloning bias towards either methylated or unmethylated DNA. Potentially complex information is simplified into a digital output, allowing counting of methylated and unmethylated alleles and providing an overall picture of methylation at the given locus. Downstream sequencing is minimised as dMS-HRM acts as a screen to select only methylated clones for further analysis.

No MeSH data available.


Related in: MedlinePlus