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Vol. 93, Issue 18, 9821-9826, September 3, 1996 (DNA methylation / tumor suppressor
genes / p16 / p15)
* Oncology Center and Communicated by Victor A. McKusick, Johns Hopkins Hospital,
Baltimore, MD, June 3, 1996 (received for review April 3, 1996)
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.
0027-8424/96/939821-6/0
Medical Sciences
Methylation-specific PCR: A novel PCR assay for methylation
status of CpG islands
,
Department of Medicine, The Johns
Hopkins Medical Institutions, 424 North Bond Street, Baltimore, MD
21231
To whom reprint requests should be addressed.
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