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The use of multiple displacement amplified DNA as a control for methylation specific PCR, pyrosequencing, bisulfite sequencing and methylation-sensitive restriction enzyme PCR.

Hughes S, Jones JL - BMC Mol. Biol. (2007)

Bottom Line: Methylation is of particular interest because of its role in gene silencing in many pathological conditions.CpG methylation can be measured using a wide range of techniques, including methylation-specific (MS) PCR, pyrosequencing (PSQ), bisulfite sequencing (BS) and methylation-sensitive restriction enzyme (MSRE) PCR.To address this problem, we have developed an approach that employs multiple displacement based whole genome amplification (WGA) with or without SssI-methylase treatment to generate CpG methylated and CpG unmethylated DNA, respectively, that come from the same source DNA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Tumour Biology Laboratory, John Vane Science Centre, Cancer Research UK Clincial Centre, Queen Mary's School of Medicine and Dentistry, UK. simon.hughes@cancer.org.uk

ABSTRACT

Background: Genomic DNA methylation affects approximately 1% of DNA bases in humans, with the most common event being the addition of a methyl group to the cytosine residue present in the CpG (cytosine-guanine) dinucleotide. Methylation is of particular interest because of its role in gene silencing in many pathological conditions. CpG methylation can be measured using a wide range of techniques, including methylation-specific (MS) PCR, pyrosequencing (PSQ), bisulfite sequencing (BS) and methylation-sensitive restriction enzyme (MSRE) PCR. However, although it is possible to utilise these methods to measure CpG methylation, optimisation of the assays can be complicated due to the absence of suitable control DNA samples.

Results: To address this problem, we have developed an approach that employs multiple displacement based whole genome amplification (WGA) with or without SssI-methylase treatment to generate CpG methylated and CpG unmethylated DNA, respectively, that come from the same source DNA.

Conclusion: Using these alternately methylated DNA samples, we have been able to develop and optimise reliable MS-PCR, PSQ, BS and MRSE-PCR assays for CpG methylation detection, which would otherwise not have been possible, or at least have been significantly more difficult.

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Methylation-Sensitive Restriction Enzyme PCR for MMP-1 and MMP-3. a) PCR using primers spanning the restriction site for MMP-1 and MMP-3 gave a PCR product with mDNA but not with uDNA. In contrast, undigested samples gave PCR products for both mDNA and uDNA. b) PCR using digested DNA from MDA-MB231 (231), MDA-MB468 (468) and HFFF2 identified that the CpG motif is methylated for all three cell lines in the MMP-3 amplicon, but only for MDA-MB468 (468) and HFFF2 for the MMP-1 amplicon, with the MDA-MB231 (231) being unmethylated. However, the undigested DNA gave a PCR product with all three cells lines.
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Figure 4: Methylation-Sensitive Restriction Enzyme PCR for MMP-1 and MMP-3. a) PCR using primers spanning the restriction site for MMP-1 and MMP-3 gave a PCR product with mDNA but not with uDNA. In contrast, undigested samples gave PCR products for both mDNA and uDNA. b) PCR using digested DNA from MDA-MB231 (231), MDA-MB468 (468) and HFFF2 identified that the CpG motif is methylated for all three cell lines in the MMP-3 amplicon, but only for MDA-MB468 (468) and HFFF2 for the MMP-1 amplicon, with the MDA-MB231 (231) being unmethylated. However, the undigested DNA gave a PCR product with all three cells lines.

Mentions: The results of the MSRE-PCR using mDNA and uDNA are shown in Figure 4a. The promoter regions of both MMP-1 and MMP-3 have a low proportion of CpG dinucleotides. Associated with some of these motifs are recognition sites for restriction enzymes that are sensitive to CpG methylation (e.g. HpyCH4IV, HpaII, SsiI), whereby when present methylation blocks the enzymes from cutting. Digestion of uDNA with HpyCH4IV resulted in cutting of DNA at unmethylated CpG motifs, however, mDNA that possesses methylated CpG motifs remained intact. Subsequent PCR, using primers spanning the restriction site, gave a PCR product with mDNA, indicating CpG methylation and protection, but not with uDNA, showing absence of CpG methylation and sensitivity to digestion. When HFFF2, MDA-MB231 and MDA-MB468 cell line DNAs were subjected to HpyCH4IV digestion and MMP-1 and MMP-3 PCR the results demonstrated that the CpG site in the MMP-1 amplicon was methylated in HFFF2 and MDA-MB468, but unmethylated in MDA-MB231 (Figure 4b). The observations for MMP-3 indicated that the CpG site is methylated in all three cell lines (Figure 4b) as all gave a PCR product. Digestion negative samples gave PCR products for all DNA samples (Figure 4a and 4b), proving that the MSRE-PCR results were specific for detection of methylation status and not a failure in the amplification reaction.


The use of multiple displacement amplified DNA as a control for methylation specific PCR, pyrosequencing, bisulfite sequencing and methylation-sensitive restriction enzyme PCR.

Hughes S, Jones JL - BMC Mol. Biol. (2007)

Methylation-Sensitive Restriction Enzyme PCR for MMP-1 and MMP-3. a) PCR using primers spanning the restriction site for MMP-1 and MMP-3 gave a PCR product with mDNA but not with uDNA. In contrast, undigested samples gave PCR products for both mDNA and uDNA. b) PCR using digested DNA from MDA-MB231 (231), MDA-MB468 (468) and HFFF2 identified that the CpG motif is methylated for all three cell lines in the MMP-3 amplicon, but only for MDA-MB468 (468) and HFFF2 for the MMP-1 amplicon, with the MDA-MB231 (231) being unmethylated. However, the undigested DNA gave a PCR product with all three cells lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175516&req=5

Figure 4: Methylation-Sensitive Restriction Enzyme PCR for MMP-1 and MMP-3. a) PCR using primers spanning the restriction site for MMP-1 and MMP-3 gave a PCR product with mDNA but not with uDNA. In contrast, undigested samples gave PCR products for both mDNA and uDNA. b) PCR using digested DNA from MDA-MB231 (231), MDA-MB468 (468) and HFFF2 identified that the CpG motif is methylated for all three cell lines in the MMP-3 amplicon, but only for MDA-MB468 (468) and HFFF2 for the MMP-1 amplicon, with the MDA-MB231 (231) being unmethylated. However, the undigested DNA gave a PCR product with all three cells lines.
Mentions: The results of the MSRE-PCR using mDNA and uDNA are shown in Figure 4a. The promoter regions of both MMP-1 and MMP-3 have a low proportion of CpG dinucleotides. Associated with some of these motifs are recognition sites for restriction enzymes that are sensitive to CpG methylation (e.g. HpyCH4IV, HpaII, SsiI), whereby when present methylation blocks the enzymes from cutting. Digestion of uDNA with HpyCH4IV resulted in cutting of DNA at unmethylated CpG motifs, however, mDNA that possesses methylated CpG motifs remained intact. Subsequent PCR, using primers spanning the restriction site, gave a PCR product with mDNA, indicating CpG methylation and protection, but not with uDNA, showing absence of CpG methylation and sensitivity to digestion. When HFFF2, MDA-MB231 and MDA-MB468 cell line DNAs were subjected to HpyCH4IV digestion and MMP-1 and MMP-3 PCR the results demonstrated that the CpG site in the MMP-1 amplicon was methylated in HFFF2 and MDA-MB468, but unmethylated in MDA-MB231 (Figure 4b). The observations for MMP-3 indicated that the CpG site is methylated in all three cell lines (Figure 4b) as all gave a PCR product. Digestion negative samples gave PCR products for all DNA samples (Figure 4a and 4b), proving that the MSRE-PCR results were specific for detection of methylation status and not a failure in the amplification reaction.

Bottom Line: Methylation is of particular interest because of its role in gene silencing in many pathological conditions.CpG methylation can be measured using a wide range of techniques, including methylation-specific (MS) PCR, pyrosequencing (PSQ), bisulfite sequencing (BS) and methylation-sensitive restriction enzyme (MSRE) PCR.To address this problem, we have developed an approach that employs multiple displacement based whole genome amplification (WGA) with or without SssI-methylase treatment to generate CpG methylated and CpG unmethylated DNA, respectively, that come from the same source DNA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Tumour Biology Laboratory, John Vane Science Centre, Cancer Research UK Clincial Centre, Queen Mary's School of Medicine and Dentistry, UK. simon.hughes@cancer.org.uk

ABSTRACT

Background: Genomic DNA methylation affects approximately 1% of DNA bases in humans, with the most common event being the addition of a methyl group to the cytosine residue present in the CpG (cytosine-guanine) dinucleotide. Methylation is of particular interest because of its role in gene silencing in many pathological conditions. CpG methylation can be measured using a wide range of techniques, including methylation-specific (MS) PCR, pyrosequencing (PSQ), bisulfite sequencing (BS) and methylation-sensitive restriction enzyme (MSRE) PCR. However, although it is possible to utilise these methods to measure CpG methylation, optimisation of the assays can be complicated due to the absence of suitable control DNA samples.

Results: To address this problem, we have developed an approach that employs multiple displacement based whole genome amplification (WGA) with or without SssI-methylase treatment to generate CpG methylated and CpG unmethylated DNA, respectively, that come from the same source DNA.

Conclusion: Using these alternately methylated DNA samples, we have been able to develop and optimise reliable MS-PCR, PSQ, BS and MRSE-PCR assays for CpG methylation detection, which would otherwise not have been possible, or at least have been significantly more difficult.

Show MeSH
Related in: MedlinePlus