RAPID QUANTITATION OF METHYLATION DIFFERENCES AT SPECIFIC SITES USINGMETHYLATION-SENSITIVE SINGLE NUCLEOTIDE PRIMER EXTENSION (MS-SNUPE)

We have developed a rapid quantitative method (Ms-SNuPE) for assessing methylation differences at specific CpG sites based on bisulfite trea tment of DNA followed by single nucleotide primer extension. Genomic D NA was first reacted with sodium bisulfite to convert unmethylated cyt osine to uracil while leaving 5-methylcytosine unchanged. Amplificatio n of the desired target sequence was then performed using PCR primers specific for bisulfite-converted DMA and the resulting product isolate d and used as a template for methylation analysis at the CpG site(s) o f interest. This methylation-sensitive technique has several advantage s over existing methods used for detection of methylation changes beca use small amounts of DNA can be analyzed including microdissected path ology sections and it avoids utilization of restriction enzymes for de termining the methylation status at CpG sites.