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A novel method to quantify local CpG methylation density by regional methylation elongation assay on microarray.

Zhang D, Wang Y, Bai Y, Ge Q, Qiao Y, Luo J, Jia C, Lu Z - BMC Genomics (2008)

Bottom Line: Indeed, difference of site-specific methylation may also lead to a change of methylation density in many cases, and it has been found that the density of methylation is more important than methylation of single CpG site for gene silencing.This technique allows for quantitative analysis of regional methylation density, which is the representative of all allelic methylation patterns in the sample.The results show that this technique has the characteristics of simplicity, rapidness, specificity and high-throughput.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China. zdd_7597@seu.edu.cn

ABSTRACT

Background: DNA methylation based techniques are important tools in both clinical diagnostics and therapeutics. But most of these methods only analyze a few CpG sites in a target region. Indeed, difference of site-specific methylation may also lead to a change of methylation density in many cases, and it has been found that the density of methylation is more important than methylation of single CpG site for gene silencing.

Results: We have developed a novel approach for quantitative analysis of CpG methylation density on the basis of microarray-based hybridization and incorporation of Cy5-dCTP into the Cy3 labeled target DNA by using Taq DNA Polymerase on microarray. The quantification is achieved by measuring Cy5/Cy3 signal ratio which is proportional to methylation density. This methylation-sensitive technique, termed RMEAM (regional methylation elongation assay on microarray), provides several advantages over existing methods used for methylation analysis. It can determine an exact methylation density of the given region, and has potential of high throughput. We demonstrate a use of this method in determining the methylation density of the promoter region of the tumor-related gene MLH1, TERT and MGMT in colorectal carcinoma patients.

Conclusion: This technique allows for quantitative analysis of regional methylation density, which is the representative of all allelic methylation patterns in the sample. The results show that this technique has the characteristics of simplicity, rapidness, specificity and high-throughput.

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Related in: MedlinePlus

A: Fully-methylated sequences of the 5' untranslated region of the positive control (fully-methylated APC allele from clone), MLH1, TERT and MGMT gene are displayed. The strand shown was amplified using the primers (green color) which are specific for bisulfite-modified DNA without CpG dinucleotides. B: The nucleotide sequences of probes, the length of targets and the number of CpG sites are shown.
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Figure 2: A: Fully-methylated sequences of the 5' untranslated region of the positive control (fully-methylated APC allele from clone), MLH1, TERT and MGMT gene are displayed. The strand shown was amplified using the primers (green color) which are specific for bisulfite-modified DNA without CpG dinucleotides. B: The nucleotide sequences of probes, the length of targets and the number of CpG sites are shown.

Mentions: We determined the feasibility of this RMEAM strategy by assessing the methylation density of CpG islands located in the promoter regions of MLH1, TERT and MGMT (Fig. 2A), and it has been proved that the mathylation of MLH1, TERT and MGMT are associated with carcinogenesis of colorectal tumor. A group of forty-arrayed oligonucleotides (Five rows of probes which have eight replicated spots; See Fig. 2B and Fig. 3A) were designed to hybridize with the labeled PCR products. The number of CpG sites in positive control (APC), MLH1, TERT and MGMT were 16, 23, 27 and 28 respectively (Fig. 2A). Fully-methylated and non-methylated DNA targets amplified from clones of these three genes were used to test the accuracy and reproducibility of the RMEAM method. A 182-bp fragment of the MLH1 promoter region, a 224-bp fragment of the TERT promoter region and a 289-bp fragment of the MGMT promoter region were amplified by triplex PCR using primers that were specific for bisulfite-converted DNAs (Fig. 2B). The fully-methylated alleles generated in this way had 100% unconverted cytosine in the test CpG sites, whereas the non-methylated alleles had all cytosine residues converted into thymine in the amplified DNA.


A novel method to quantify local CpG methylation density by regional methylation elongation assay on microarray.

Zhang D, Wang Y, Bai Y, Ge Q, Qiao Y, Luo J, Jia C, Lu Z - BMC Genomics (2008)

A: Fully-methylated sequences of the 5' untranslated region of the positive control (fully-methylated APC allele from clone), MLH1, TERT and MGMT gene are displayed. The strand shown was amplified using the primers (green color) which are specific for bisulfite-modified DNA without CpG dinucleotides. B: The nucleotide sequences of probes, the length of targets and the number of CpG sites are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2262069&req=5

Figure 2: A: Fully-methylated sequences of the 5' untranslated region of the positive control (fully-methylated APC allele from clone), MLH1, TERT and MGMT gene are displayed. The strand shown was amplified using the primers (green color) which are specific for bisulfite-modified DNA without CpG dinucleotides. B: The nucleotide sequences of probes, the length of targets and the number of CpG sites are shown.
Mentions: We determined the feasibility of this RMEAM strategy by assessing the methylation density of CpG islands located in the promoter regions of MLH1, TERT and MGMT (Fig. 2A), and it has been proved that the mathylation of MLH1, TERT and MGMT are associated with carcinogenesis of colorectal tumor. A group of forty-arrayed oligonucleotides (Five rows of probes which have eight replicated spots; See Fig. 2B and Fig. 3A) were designed to hybridize with the labeled PCR products. The number of CpG sites in positive control (APC), MLH1, TERT and MGMT were 16, 23, 27 and 28 respectively (Fig. 2A). Fully-methylated and non-methylated DNA targets amplified from clones of these three genes were used to test the accuracy and reproducibility of the RMEAM method. A 182-bp fragment of the MLH1 promoter region, a 224-bp fragment of the TERT promoter region and a 289-bp fragment of the MGMT promoter region were amplified by triplex PCR using primers that were specific for bisulfite-converted DNAs (Fig. 2B). The fully-methylated alleles generated in this way had 100% unconverted cytosine in the test CpG sites, whereas the non-methylated alleles had all cytosine residues converted into thymine in the amplified DNA.

Bottom Line: Indeed, difference of site-specific methylation may also lead to a change of methylation density in many cases, and it has been found that the density of methylation is more important than methylation of single CpG site for gene silencing.This technique allows for quantitative analysis of regional methylation density, which is the representative of all allelic methylation patterns in the sample.The results show that this technique has the characteristics of simplicity, rapidness, specificity and high-throughput.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China. zdd_7597@seu.edu.cn

ABSTRACT

Background: DNA methylation based techniques are important tools in both clinical diagnostics and therapeutics. But most of these methods only analyze a few CpG sites in a target region. Indeed, difference of site-specific methylation may also lead to a change of methylation density in many cases, and it has been found that the density of methylation is more important than methylation of single CpG site for gene silencing.

Results: We have developed a novel approach for quantitative analysis of CpG methylation density on the basis of microarray-based hybridization and incorporation of Cy5-dCTP into the Cy3 labeled target DNA by using Taq DNA Polymerase on microarray. The quantification is achieved by measuring Cy5/Cy3 signal ratio which is proportional to methylation density. This methylation-sensitive technique, termed RMEAM (regional methylation elongation assay on microarray), provides several advantages over existing methods used for methylation analysis. It can determine an exact methylation density of the given region, and has potential of high throughput. We demonstrate a use of this method in determining the methylation density of the promoter region of the tumor-related gene MLH1, TERT and MGMT in colorectal carcinoma patients.

Conclusion: This technique allows for quantitative analysis of regional methylation density, which is the representative of all allelic methylation patterns in the sample. The results show that this technique has the characteristics of simplicity, rapidness, specificity and high-throughput.

Show MeSH
Related in: MedlinePlus