Microarray-based Ms-SNuPE: near-quantitative analysis for a high-throughput DNA methylation.
Aberrant DNA methylation of CpG site in the gene promoter region has been confirmed to be closely associated with carcinogenesis. In the present study, a microarray-based methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) for parallel detecting changes of DNA methylation in cancer was developed. After modification by sodium sulfite, the unmethylated cytosine in the genomic DNA is converted to uracil while leaving the 5-methylcytosine unchanged, which can be detected by bifunctional primer carrying a unique sequence tag in addition to a locus-specific sequence. Because each locus has a distinct tag, the detecting reactions can be performed in a highly multiplexed fashion and the resulting product then be hybridized to the reverse complements of the sequence tags arrayed on a glass slide for methylation analysis. The calibration curves with the correlation coefficient >0.97 were established, which suggested that the method could be used in near-quantitative DNA methylation analysis. Two breast tumor-related genes (E-cad and p16) are successfully analyzed by two group primers (22 primers total), and the results are compatible with that of methylation-specific PCR (MSP). Our research proved that the method is simple and inexpensive, and could be applied as a high-throughput tool to quantitatively determine methylation status of the investigated genes.
Wu
Zhixiang
Z
State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China.
Luo
Junfeng
J
Ge
Qinyu
Q
Lu
Zuhong
Z
D018899:CpG Islands; D019175:DNA Methylation; D005189:False Positive Reactions; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results