High-throughput method for detecting DNA methylation.
Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in cancer. Detection of promoter hypermethylation of cancer-related gene may be useful for cancer diagnosis or the detection of recurrence. However, most of the studies have focused on a single gene only and gave little information about the concurrent methylation status of multiple genes. In this study, we attempted to develop a microarray method coupled with linker-PCR for detecting methylation status of multiple genes in the tumor tissue. A series of synthesized oligonucleotides were synthesised and purified to completely match with 16 investigated targets. Then they were immobilized on the aldehyde-coated glass slide to fabricate a DNA microarray for detecting methylation status of these genes. The results indicated that these genes were all methylated in the positive control. However, no methylated was found in these genes for the negative control. Only p16 and p15 genes were methylated in investigated genes for the gastric tumor tissue, whereas others were not methylated. The above results were validated by bisulfite DNA sequencing. Our experiments successfully demonstrated that the DNA microarray could be applied as a high-throughput tool to determine methylation status of the investigated genes.
Hou
Peng
P
Chien-Shiung Wu Laboratory, Department of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China.
Ji
Meiju
M
Li
Song
S
He
Nongyue
N
Lu
Zuhong
Z
D001331:Automation; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000235:genetics; D018899:CpG Islands; D050762:Cyclin-Dependent Kinase Inhibitor p15; D004247:DNA; D019175:DNA Methylation; D019942:Genes, p16; D005821:Genetic Techniques; D005898:Glass; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D009841:Oligonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000378:metabolism; D013447:Sulfites / Q000737:chemistry; D013997:Time Factors; D025521:Tumor Suppressor Proteins / Q000235:genetics
C493966:CDKN2B protein, human; D018797:Cell Cycle Proteins; D050762:Cyclin-Dependent Kinase Inhibitor p15; D009841:Oligonucleotides; D013447:Sulfites; D025521:Tumor Suppressor Proteins; D004247:DNA