Identification of DNA methylation differences during tumorigenesis by methylation-sensitive arbitrarily primed polymerase chain reaction.
The ability to detect methylation changes associated with oncogenic transformation is of critical importance in understanding how DNA methylation may contribute to tumorigenesis. We have developed a simple and reproducible fingerprinting method called methylation-sensitive arbitrarily primed polymerase chain reaction (AP-PCR) to screen for DNA methylation changes. This technique relies on digesting genomic DNA with methylation-sensitive and -insensitive restriction enzymes (e.g., HpaII and MspI) prior to AP-PCR amplification. Matched normal and tumor DNAs were compared to identify differential methylation. After the PCR products were resolved on high-resolution polyacrylamide gels, regions of genomic DNA that showed hypo- and hypermethylation associated with tumors were detected. These fragments were then isolated, cloned, and sequenced. Novel CpG islands were 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.
Liang
Gangning
G
Department of Biochemistry and Molecular Biology, Urologic Cancer Research Laboratory, USC/Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA. gliang@hsc.usc.edu
Gonzalgo
Mark L
ML
Salem
Carol
C
Jones
Peter A
PA
D015139:Blotting, Southern / Q000379:methods; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D003110:Colonic Neoplasms; D018899:CpG Islands; D019175:DNA Methylation; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D004273:DNA, Neoplasm