Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.
Precise mapping of DNA methylation patterns in CpG islands has become essential for understanding diverse biological processes such as the regulation of imprinted genes, X chromosome inactivation, and tumor suppressor gene silencing in human cancer. We describe a new method, MSP (methylation-specific PCR), which can rapidly assess the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of methylation-sensitive restriction enzymes. This assay entails initial modification of DNA by sodium bisulfite, converting all unmethylated, but not methylated, cytosines to uracil, and subsequent amplification with primers specific for methylated versus unmethylated DNA. MSP requires only small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. MSP eliminates the false positive results inherent to previous PCR-based approaches which relied on differential restriction enzyme cleavage to distinguish methylated from unmethylated DNA. In this study, we demonstrate the use of MSP to identify promoter region hypermethylation changes associated with transcriptional inactivation in four important tumor suppressor genes (p16, p15, E-cadherin, and von Hippel-Lindau) in human cancer.
Herman
J G
JG
Oncology Center, Johns Hopkins Medical Institutions, Baltimore, MD 21231, USA.
Graff
J R
JR
Myöhänen
S
S
Nelkin
B D
BD
Baylin
S B
SB
D016375:Antisense Elements (Genetics) / Q000737:chemistry; D001483:Base Sequence; D015820:Cadherins / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics; D018797:Cell Cycle Proteins; D002460:Cell Line; D018899:CpG Islands; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D017931:DNA Primers / Q000138:chemical synthesis; D016147:Genes, Tumor Suppressor; D006801:Humans; D008025:Ligases; D008745:Methylation; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011506:Proteins / Q000235:genetics; D015183:Restriction Mapping; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein
D016375:Antisense Elements (Genetics); C493966:CDKN2B protein, human; D015820:Cadherins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D017931:DNA Primers; D011506:Proteins; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D008025:Ligases; C497446:VHL protein, human