Identification and characterization of differentially methylated regions of genomic DNA by methylation-sensitive arbitrarily primed PCR.
We have developed a simple and reproducible fingerprinting method for screening the genome for regions of DNA that have altered patterns of DNA methylation associated with oncogenic transformation. Restriction enzymes with different sensitivities to cytosine methylation in their recognition sites were used to digest genomic DNAs from primary tumors, cell lines, and normal tissues prior to arbitrarily primed PCR amplification. Fragments that showed differential methylation were cloned and sequenced after resolving the PCR products on high-resolution polyacrylamide gels. The cloned fragments were then used as probes for Southern analysis to confirm differential methylation of these regions in colon tissues and cell lines. Forty-four DNA fragments associated with a total of five different regions of genomic DNA containing methylation sites were detected in 10 matched sets of normal and tumor colon DNAs and 7 colon cancer cell lines. A novel CpG island was also isolated that was found to be frequently hypermethylated in bladder and colon tumors. We have demonstrated that this technique is a rapid and efficient method that can be used to screen for altered methylation patterns in genomic DNA and to isolate specific sequences associated with these changes.
Gonzalgo
M L
ML
Department of Biochemistry and Molecular Biology, University of Southern California/Norris Comprehensive Cancer Center, Los Angeles 90033, USA.
Liang
G
G
Spruck
C H
CH
Zingg
J M
JM
Rideout
W M
WM
Jones
P A
PA
D015139:Blotting, Southern; D003110:Colonic Neoplasms / Q000235:genetics*; D016172:DNA Fingerprinting / Q000379:methods*; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000235:genetics*
D004273:DNA, Neoplasm