Rapid quantitation of methylation differences at specific sites using methylation-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 treatment of DNA followed by single nucleotide primer extension. Genomic DNA was first reacted with sodium bisulfite to convert unmethylated cytosine to uracil while leaving 5-methylcytosine unchanged. Amplification of the desired target sequence was then performed using PCR primers specific for bisulfite-converted DNA and the resulting product isolated and used as a template for methylation analysis at the CpG site(s) of interest. This methylation-sensitive technique has several advantages over existing methods used for detection of methylation changes because small amounts of DNA can be analyzed including microdissected pathology sections and it avoids utilization of restriction enzymes for determining the methylation status at CpG sites.
Gonzalgo
M L
ML
Department of Biochemistry and Molecular Biology, Urologic Cancer Research Laboratory USC/Norris Comprehensive Cancer Center, University of Southern California, School of Medicine, Los Angeles, CA 90033, USA.
Jones
P A
PA
D004247:DNA / Q000737:chemistry*; D019175:DNA Methylation; D017931:DNA Primers; D015226:Dinucleoside Phosphates / Q000737:chemistry; D005821:Genetic Techniques; D006801:Humans; D007202:Indicators and Reagents; D008832:Microchemistry / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods; D015183:Restriction Mapping; D013431:Sulfates; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms
D004247:DNA; D017931:DNA Primers; D015226:Dinucleoside Phosphates; D007202:Indicators and Reagents; D013431:Sulfates; C012036:sodium sulfate; C015772:CpG dinucleotide