In situ bisulfite modification of membrane-immobilized DNA for multiple methylation analysis.
Increasing interest in DNA methylation has resulted in the recent development of array-based methods. However, these method require complex sample treatment processes, such as bisulfite modification in the liquid state of every sample. Here, we describe a microarray-based technology for interrogating DNA methylation status of multiple DNA samples at the same time, in which the DNA samples are first dotted on membranes and then treated with bisulfite directly. In this assay, plasmid pUC19 DNA is immobilized on nylon membranes and soaked in bisulfite reaction mixtures for 16h to convert unmethylated Cs to Us, and methylated Cs remain unchanged. The probes for detecting the methylation patterns of CpG sites are hybridized with the converted DNA dotted on the membranes, and the signals are revealed by chemiluminescence of DIG-labeled probes. The experiments show that this method can simplify the experimental processes and increase the efficiency of the bisulfite treatment. This new method could be used as a convenient tool for detecting the methylation status of multiple genes for a large number of samples in the future.
Wang
Yan
Y
The State Key Laboratory of Bioelectronics, Department of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China.
Zheng
Wenli
W
Luo
Junfeng
J
Zhang
Dingdong
D
Zuhong
Lu
L
D019411:Clinical Laboratory Techniques; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation; D015257:DNA-Cytosine Methylases / Q000235:genetics; D044127:Epigenesis, Genetic; D005821:Genetic Techniques; D008566:Membranes / Q000737:chemistry*; D009693:Nucleic Acid Hybridization / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D013447:Sulfites / Q000737:chemistry*
D013447:Sulfites; D015257:DNA-Cytosine Methylases; C042345:hydrogen sulfite