Identification of differentially methylated sites within unmethylated DNA domains in normal and cancer cells.
Altered DNA methylation has been linked to neoplastic cell transformation and is a hallmark of cancer progression. Therefore, the screening for differentially methylated sequences as tumor biomarkers has a significant implication in the clinical setting. To determine the cancer-linked alterations in DNA methylation pattern, we have applied an endonuclease, McrBC, to the existing methylation-sensitive arbitrarily primed polymerase chain reaction (msAP-PCR) method and developed McrBC-msAP-PCR. This modified approach allows detection of differentially methylated sites within unmethylated DNA domains enriched by regulatory sequences and CpG islands. In this method, we used digestion of DNA with the McrBC methylation-sensitive endonuclease to selectively exclude the methylated fraction of DNA, which comprises interspersed and tandem-repeated sequences and exons other than first exons, from analysis. The subsequent digestion of unmethylated DNA fragments with SmaI and HpaII methylation-sensitive restriction endonucleases followed by AP-PCR amplification resulted in the detection of unknown unique sequences associated with cancer-linked methylation changes in genomic DNA. Hypermethylation and hypomethylation are visualized by the increase or decrease in the band intensity of DNA fingerprints. By using this technique, we were able to differentiate clearly, identify, and characterize a number of novel unique DNA sequences with differentially methylated sites in normal and breast cancer cell lines and in normal and rat tumor liver tissues.
Tryndyak
Volodymyr
V
Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, USA.
Kovalchuk
Olga
O
Pogribny
Igor P
IP
D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D018899:CpG Islands / Q000235:genetics*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D019003:Deoxyribonuclease HpaII / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D051381:Rats; D017422:Sequence Analysis, DNA / Q000379:methods
D004273:DNA, Neoplasm; D004247:DNA; D004720:Endonucleases; D004262:DNA Restriction Enzymes; D019003:Deoxyribonuclease HpaII; C120301:McrBC endonuclease