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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

Outline for the analysis of gene promoter methylation density.
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Figure 1: Outline for the analysis of gene promoter methylation density.

Mentions: Figure 1 outlines the RMEAM (regional methylation elongation assay on microarray) strategy for DNA methylation density analysis. Test DNA samples are bisulfite-modified, PCR amplified products which contain pools of DNA fragments with altered nucleotide sequences due to their differential methylation status. As shown, the unmethylated allele of a given DNA sequence is expected to have the unmethylated cytosine of the test CpG sites converted to thymine, whereas these CpG sequences remain unchanged in the methylated allele. The information on cytosine methylation can now be converted into sequence information. The region of interest is amplified with primers that are specific for bisulfite-modified DNA without CpG dinucleotides in the sequence, which ensures that the PCR amplification is independent of the original methylation status. Thus, the number of CpG dinucleotides remaining in the amplified region would reflect original methylation status. Subsequently, the forward primer was modified with -NH2 at 5' end, and then fixed on the aldehyde coating glass as probe leaving 3'-OH free. Target DNA is then hybridized to arrayed oligonucleotide probes which are specifically designed to match the reverse strands. For quantification of CpG dinucleotides, enzymatic elongation was carried out with dNTPs (dATP, dGTP, dTTP, dCTP, Cy5-dCTP), PCR buffer, Mg2+ and Taq enzyme on microarray. The incorporation of Cy5-dCTP into the DNA is proportional to the number of methylated CpG sites originally present in this DNA region. To minimize the background signal and solve the problem that a variable quantity of DNA has different fluorescence intensities in every reaction tube, we devised a smart way to accurately standardize between samples. For this step, we modified Cy3 at the 5'end of reverse primers and the Cy3 labeled reverse strands were hybridized to the oligonucleotide probes. Because the number of Cy3 is identical for every PCR product, the Cy3 signal can be used as an internal control to standardize the amount of DNA that is finally analyzed. The results are expressed as fluorescence intensity ratio (Cy5/Cy3). By use of the mixture of fully-methylated and non-methylated alleles as standards, the ratios can be converted into percentage values, and thus, the average methylation density of the amplified region is determined. However, considering standardization of Cy3 and Cy5 fluorescence intensity as well as quality control, we use a positive control (fully-methylated APC allele from clone) just like the housekeeping gene of cDNA microarray and a negative control which removes the influence of cross-hybridization. This positive control has definite number of CpG sites and merges the PCR products before the hybridization. Thus, the fluorescence intensity ratios (Cy5sam*Cy3pos)/(Cy5pos*Cy3sam) will be proportional to the methylation density in the original sequence (sam: sample; pos: positive control).


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)

Outline for the analysis of gene promoter methylation density.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Outline for the analysis of gene promoter methylation density.
Mentions: Figure 1 outlines the RMEAM (regional methylation elongation assay on microarray) strategy for DNA methylation density analysis. Test DNA samples are bisulfite-modified, PCR amplified products which contain pools of DNA fragments with altered nucleotide sequences due to their differential methylation status. As shown, the unmethylated allele of a given DNA sequence is expected to have the unmethylated cytosine of the test CpG sites converted to thymine, whereas these CpG sequences remain unchanged in the methylated allele. The information on cytosine methylation can now be converted into sequence information. The region of interest is amplified with primers that are specific for bisulfite-modified DNA without CpG dinucleotides in the sequence, which ensures that the PCR amplification is independent of the original methylation status. Thus, the number of CpG dinucleotides remaining in the amplified region would reflect original methylation status. Subsequently, the forward primer was modified with -NH2 at 5' end, and then fixed on the aldehyde coating glass as probe leaving 3'-OH free. Target DNA is then hybridized to arrayed oligonucleotide probes which are specifically designed to match the reverse strands. For quantification of CpG dinucleotides, enzymatic elongation was carried out with dNTPs (dATP, dGTP, dTTP, dCTP, Cy5-dCTP), PCR buffer, Mg2+ and Taq enzyme on microarray. The incorporation of Cy5-dCTP into the DNA is proportional to the number of methylated CpG sites originally present in this DNA region. To minimize the background signal and solve the problem that a variable quantity of DNA has different fluorescence intensities in every reaction tube, we devised a smart way to accurately standardize between samples. For this step, we modified Cy3 at the 5'end of reverse primers and the Cy3 labeled reverse strands were hybridized to the oligonucleotide probes. Because the number of Cy3 is identical for every PCR product, the Cy3 signal can be used as an internal control to standardize the amount of DNA that is finally analyzed. The results are expressed as fluorescence intensity ratio (Cy5/Cy3). By use of the mixture of fully-methylated and non-methylated alleles as standards, the ratios can be converted into percentage values, and thus, the average methylation density of the amplified region is determined. However, considering standardization of Cy3 and Cy5 fluorescence intensity as well as quality control, we use a positive control (fully-methylated APC allele from clone) just like the housekeeping gene of cDNA microarray and a negative control which removes the influence of cross-hybridization. This positive control has definite number of CpG sites and merges the PCR products before the hybridization. Thus, the fluorescence intensity ratios (Cy5sam*Cy3pos)/(Cy5pos*Cy3sam) will be proportional to the methylation density in the original sequence (sam: sample; pos: positive control).

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