Colorimetric silver detection of methylation using DNA microarray coupled with linker-PCR.
BACKGROUND
Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in cancer. Detection of promoter hypermethylation of cancer-related genes may be useful for cancer diagnosis or the detection of recurrence. Recently, several DNA microarray methods have been developed to detect the methylation status of the multiple genes. However, the microarrays are currently detected in fluorescence using a sophisticated laser-based scanner. These methods are limited by their lower sensitivity.


METHODS
We present a sensitive colorimetric silver method coupled with linker-PCR to detect methylation status of four genes, including p16, E-cadherin, VHL, and hMLH1. The signal generated with this method results from the precipitation of silver onto nanogold particles bound to streptavidin used to detect biotinylated DNA.


RESULTS
We show that p16, E-cadherin, VHL, and hMLH1 were all methylated in the positive control. However, no methylation was found in these genes for the negative control. P16 gene was only methylated in the sample, whereas others were not methylated. Furthermore, as little as 0.1 fmol of target DNA amplicons could be detected on the arrays. The results were further validated by methylation-specific PCR (MSP) and bisulfite DNA sequencing.


CONCLUSIONS
The colorimetric silver detection of DNA microarray could be used as an inexpensive, useful, and sensitive tool to detect methylation status of the multiple genes in the future.
Ji
Meiju
M
Chien-Shiung Wu Laboratory, Department of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China.
Hou
Peng
P
Li
Song
S
He
Nongyue
N
Lu
Zuhong
Z
D048868:Adaptor Proteins, Signal Transducing; D015820:Cadherins / Q000235:genetics; D002352:Carrier Proteins; D003124:Colorimetry / Q000379:methods; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics; D019175:DNA Methylation; D020869:Gene Expression Profiling / Q000379:methods; D006801:Humans; D007202:Indicators and Reagents; D000070957:MutL Protein Homolog 1; D009363:Neoplasm Proteins / Q000235:genetics; D009687:Nuclear Proteins; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D012834:Silver; D025521:Tumor Suppressor Proteins / Q000235:genetics; D044767:Ubiquitin-Protein Ligases / Q000235:genetics; D051794:Von Hippel-Lindau Tumor Suppressor Protein
D048868:Adaptor Proteins, Signal Transducing; D015820:Cadherins; D002352:Carrier Proteins; D019941:Cyclin-Dependent Kinase Inhibitor p16; D007202:Indicators and Reagents; C501486:MLH1 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D025521:Tumor Suppressor Proteins; D012834:Silver; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D000070957:MutL Protein Homolog 1; C497446:VHL protein, human