Isolation and enrichment of human genomic CpG islands by methylation-sensitive mirror orientation selection.
CpG islands (CGIs) in human genomic DNA are GC-rich fragments whose aberrant methylation is associated with human disease development. In the current study, methylation-sensitive mirror orientation selection (MS-MOS) was developed to efficiently isolate and enrich unmethylated CGIs from human genomic DNA. The unmethylated CGIs prepared by the MS-MOS procedure subsequently were used to construct a CGI library. Then the sequence characteristics of cloned inserts of the library were analyzed by bioinformatics tools, and the methylation status of CGI clones was analyzed by HpaII PCR. The results showed that the MS-MOS method could be used to isolate up to 0.001% of differentially existed unmethylated DNA fragments in two complex genomic DNA. In the CGI library, 34.1% of clones had insert sequences satisfying the minimal criteria for CGIs. Excluding duplicates, 22.0% of the 80,000 clones were unique CGI clones, representing 60% of all the predicted CGIs (about 29,000) in human genomic DNA, and most or all of the CGI clones were unmethylated in human normal cell DNA based on the HpaII PCR analysis results of randomly selected CGI clones. In conclusion, MS-MOS was an efficient way to isolate and enrich human genomic CGIs. The method has powerful potential application in the comprehensive identification of aberrantly methylated CGIs associated with human tumorigenesis to improve understanding of the epigenetic mechanisms involved.
Huang
Qing
Q
Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing 400038, People's Republic of China.
Baum
Larry
L
Huang
Jun-Fu
JF
You
Jian-Ping
JP
Wang
Feng
F
Wang
Jue
J
Zheng
Jiang
J
Yan
Xiao-Chu
XC
Xia
Han
H
Zhao
Yu-Hui
YH
Kuang
Hong
H
Fu
Wei-Ling
WL
D018899:CpG Islands / Q000235:genetics*; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D019175:DNA Methylation; D015894:Genome, Human / Q000235:genetics*; D006801:Humans; D008297:Male
D004247:DNA