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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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Related in: MedlinePlus

Flow diagram demonstrating the steps involved in generation of differentially methylated DNA and the downstream applications of the DNA.
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Figure 1: Flow diagram demonstrating the steps involved in generation of differentially methylated DNA and the downstream applications of the DNA.

Mentions: MDA is a rolling circle amplification method, originally developed for the amplification of large circular DNA templates [12], which has been adapted for the amplification of the entire genome [13,14]. This amplification method can generate DNA strands in excess of 10 kb in length, without prior knowledge of the target template [15]. In the context of this work, MDA generates amplified DNA free of any methylation due to the absence of methylase activity for the MDA enzyme (phi29 polymerase). As a consequence the DNA generated my MDA will be unmethylated DNA (uDNA). SssI methylase has been reported to methylate the fifth position of cytosine in all CpG dinucleotides [16], thus the treatment of MDA generated DNA with M.SssI will generate CpG methylated DNA (mDNA). A flow diagram of the steps involved is displayed in Figure 1.


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)

Flow diagram demonstrating the steps involved in generation of differentially methylated DNA and the downstream applications of the DNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Flow diagram demonstrating the steps involved in generation of differentially methylated DNA and the downstream applications of the DNA.
Mentions: MDA is a rolling circle amplification method, originally developed for the amplification of large circular DNA templates [12], which has been adapted for the amplification of the entire genome [13,14]. This amplification method can generate DNA strands in excess of 10 kb in length, without prior knowledge of the target template [15]. In the context of this work, MDA generates amplified DNA free of any methylation due to the absence of methylase activity for the MDA enzyme (phi29 polymerase). As a consequence the DNA generated my MDA will be unmethylated DNA (uDNA). SssI methylase has been reported to methylate the fifth position of cytosine in all CpG dinucleotides [16], thus the treatment of MDA generated DNA with M.SssI will generate CpG methylated DNA (mDNA). A flow diagram of the steps involved is displayed in Figure 1.

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