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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 Specific PCR results for MMP-2 and BRCA-1. Two sets of primers were designed for both MMP-2 and BRCA-1, one set that would amplify only methylated DNA and a second set that would amplify only unmethylated DNA. a) Those primers that were designed to amplify methylated DNA only amplified mDNA and not uDNA or genomic DNA, whilst those primers designed to amplify unmethylated DNA only amplified uDNA and not mDNA or genomic DNA. Furthermore wild type primers were unable to amplify either uDNA or mDNA, but could amplify genomic DNA. b) When used in conjunction with cell line DNA they detected that the MMP-2 promoter is methylated for MDA-MB231 (231) and MDA-MB468 (468), but not HFFF2. However, the promoters for MDA-MB231 (231), MDA-MB468 (468) and HFFF2 were all identified as being unmethylated for BRCA-1.
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Figure 2: Methylation Specific PCR results for MMP-2 and BRCA-1. Two sets of primers were designed for both MMP-2 and BRCA-1, one set that would amplify only methylated DNA and a second set that would amplify only unmethylated DNA. a) Those primers that were designed to amplify methylated DNA only amplified mDNA and not uDNA or genomic DNA, whilst those primers designed to amplify unmethylated DNA only amplified uDNA and not mDNA or genomic DNA. Furthermore wild type primers were unable to amplify either uDNA or mDNA, but could amplify genomic DNA. b) When used in conjunction with cell line DNA they detected that the MMP-2 promoter is methylated for MDA-MB231 (231) and MDA-MB468 (468), but not HFFF2. However, the promoters for MDA-MB231 (231), MDA-MB468 (468) and HFFF2 were all identified as being unmethylated for BRCA-1.

Mentions: The use of bisulfite treated mDNA and uDNA as template for MS-PCR has allowed for the optimisation of several primer sets. Primers for MS-PCR will ideally only generate a product with either mDNA or uDNA, but not both. Typical results obtained are displayed in Figure 2a. Using MMP-2 and BRCA-1 as examples, the primers that were designed to amplify methylated DNA only amplified mDNA and not uDNA. Conversely those primers that were designed to amplify unmethylated DNA only amplified uDNA and not mDNA. None of the MS-PCR primers sets amplified untreated genomic DNA; in addition wild-type primers did not amplify the bisulfite treated mDNA or uDNA (Figure 2a). When the primers were applied to bisulfite treated DNA from the HFFF2, MDA-MB231 and MDA-MB468 cell lines, all three were demonstrated to be unmethylated for BRCA-1 (Figure 2b). The analysis of MMP-2 methylation status indicated that both MDA-MB231 and MDA-MB468 were methylated, whilst HFFF2 was unmethylated (Figure 2b).


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 Specific PCR results for MMP-2 and BRCA-1. Two sets of primers were designed for both MMP-2 and BRCA-1, one set that would amplify only methylated DNA and a second set that would amplify only unmethylated DNA. a) Those primers that were designed to amplify methylated DNA only amplified mDNA and not uDNA or genomic DNA, whilst those primers designed to amplify unmethylated DNA only amplified uDNA and not mDNA or genomic DNA. Furthermore wild type primers were unable to amplify either uDNA or mDNA, but could amplify genomic DNA. b) When used in conjunction with cell line DNA they detected that the MMP-2 promoter is methylated for MDA-MB231 (231) and MDA-MB468 (468), but not HFFF2. However, the promoters for MDA-MB231 (231), MDA-MB468 (468) and HFFF2 were all identified as being unmethylated for BRCA-1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Methylation Specific PCR results for MMP-2 and BRCA-1. Two sets of primers were designed for both MMP-2 and BRCA-1, one set that would amplify only methylated DNA and a second set that would amplify only unmethylated DNA. a) Those primers that were designed to amplify methylated DNA only amplified mDNA and not uDNA or genomic DNA, whilst those primers designed to amplify unmethylated DNA only amplified uDNA and not mDNA or genomic DNA. Furthermore wild type primers were unable to amplify either uDNA or mDNA, but could amplify genomic DNA. b) When used in conjunction with cell line DNA they detected that the MMP-2 promoter is methylated for MDA-MB231 (231) and MDA-MB468 (468), but not HFFF2. However, the promoters for MDA-MB231 (231), MDA-MB468 (468) and HFFF2 were all identified as being unmethylated for BRCA-1.
Mentions: The use of bisulfite treated mDNA and uDNA as template for MS-PCR has allowed for the optimisation of several primer sets. Primers for MS-PCR will ideally only generate a product with either mDNA or uDNA, but not both. Typical results obtained are displayed in Figure 2a. Using MMP-2 and BRCA-1 as examples, the primers that were designed to amplify methylated DNA only amplified mDNA and not uDNA. Conversely those primers that were designed to amplify unmethylated DNA only amplified uDNA and not mDNA. None of the MS-PCR primers sets amplified untreated genomic DNA; in addition wild-type primers did not amplify the bisulfite treated mDNA or uDNA (Figure 2a). When the primers were applied to bisulfite treated DNA from the HFFF2, MDA-MB231 and MDA-MB468 cell lines, all three were demonstrated to be unmethylated for BRCA-1 (Figure 2b). The analysis of MMP-2 methylation status indicated that both MDA-MB231 and MDA-MB468 were methylated, whilst HFFF2 was unmethylated (Figure 2b).

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