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


CONCLUSIONS
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.
Zhang
Dingdong
D
State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China. zdd_7597@seu.edu.cn
Wang
Yan
Y
Bai
Yunfei
Y
Ge
Qinyu
Q
Qiao
Yingjuan
Y
Luo
Junfeng
J
Jia
Chao
C
Lu
Zuhong
Z
D020121:5' Untranslated Regions; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics; D001483:Base Sequence; D002232:Carbocyanines; D015179:Colorectal Neoplasms / Q000737:chemistry / Q000235:genetics; D018899:CpG Islands; D004247:DNA / Q000737:chemistry; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000235:genetics; D045643:DNA Repair Enzymes / Q000235:genetics; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D005456:Fluorescent Dyes; D006801:Humans; D008969:Molecular Sequence Data; D000070957:MutL Protein Homolog 1; D009687:Nuclear Proteins / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D011401:Promoter Regions, Genetic; D019914:Taq Polymerase; D019098:Telomerase / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000235:genetics
D020121:5' Untranslated Regions; D048868:Adaptor Proteins, Signal Transducing; D002232:Carbocyanines; D004273:DNA, Neoplasm; D005456:Fluorescent Dyes; C501486:MLH1 protein, human; D009687:Nuclear Proteins; D025521:Tumor Suppressor Proteins; C089187:cyanine dye 3; D004247:DNA; D015254:DNA Modification Methylases; C515491:MGMT protein, human; D019914:Taq Polymerase; C509186:TERT protein, human; D019098:Telomerase; D000070957:MutL Protein Homolog 1; D045643:DNA Repair Enzymes