A rapid, quantitative, non-radioactive bisulfite-SNuPE- IP RP HPLC assay for methylation analysis at specific CpG sites.
The precise mapping and quantification of DNA methylation as an epigenetic parameter during development and in diseased tissues is of great importance for functional genomics. Here we describe a rapid, quantitative method to assess methylation levels at specific CpG sites using PCR products of bisulfite-treated genomic DNA. Using single nucleotide primer extension (SNuPE) assays in combination with ion pair reverse phase high performance liquid chromatography (IP RP HPLC) separation techniques, methylated and unmethylated CpGs can be discriminated and quantified based on the different masses and hydrophobicities of the extended primer products. The assay is linear, highly reproducible and several sites can be measured simultaneously in one reaction. It can be semi-automated and eliminates the need for cloning and sequencing of individual bisulfite PCR products.
El-Maarri
Osman
O
Institute of Experimental Haematology and Transfusion Medicine, Sigmund-Freud Strasse 25, 53105 Bonn, Germany. osman.elmaarri@ukb.uni-bonn.de
Herbiniaux
Ursula
U
Walter
Jörn
J
Oldenburg
Johannes
J
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D018899:CpG Islands; D019175:DNA Methylation; D017931:DNA Primers; D057927:Hydrophobic and Hydrophilic Interactions; D016133:Polymerase Chain Reaction; D011868:Radioisotopes; D015203:Reproducibility of Results; D013447:Sulfites / Q000737:chemistry*; D013997:Time Factors
D017931:DNA Primers; D011868:Radioisotopes; D013447:Sulfites