MethylQuant: a sensitive method for quantifying methylation of specific cytosines within the genome.
Here we present MethylQuant, a novel method that allows accurate quantification of the methylation level of a specific cytosine within a complex genome. This method relies on the well-established treatment of genomic DNA with sodium bisulfite, which converts cytosine into uracil without modifying 5-methyl cytosine. The region of interest is then PCR-amplified and quantification of the methylation status of a specific cytosine is performed by methylation-specific real-time PCR with SYBR Green I using one of the primers whose 3' end discriminates between the methylation states of this cytosine. The presence of a locked nucleic acid at the 3' end of the discriminative primer provides the specificity necessary for accurate and sensitive quantification, even when one of the methylation states is present at a level as low as 1% of the overall population. We demonstrate that accurate quantification of the methylation status of specific cytosines can be achieved in biological samples. The method is high-throughput, cost-effective, relatively simple and does not require any specific equipment other than a real-time PCR instrument.
Thomassin
Hélène
H
Institut Jacques Monod du CNRS, Universités Paris 6-7, Tour 43, 2 place Jussieu, 75251 Paris Cedex 05, France.
Kress
Clémence
C
Grange
Thierry
T
D044503:5-Methylcytosine / Q000032:analysis*; D000818:Animals; D052160:Benzothiazoles; D045744:Cell Line, Tumor; D018899:CpG Islands; D019175:DNA Methylation; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000032:analysis; D003959:Diamines; D005456:Fluorescent Dyes; D023281:Genomics / Q000379:methods; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense / Q000737:chemistry; D009930:Organic Chemicals / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D051381:Rats; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites / Q000737:chemistry
D052160:Benzothiazoles; D017931:DNA Primers; D004273:DNA, Neoplasm; D003959:Diamines; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D009930:Organic Chemicals; D011804:Quinolines; D013447:Sulfites; C477371:locked nucleic acid; C098022:SYBR Green I; D044503:5-Methylcytosine; C009279:sodium bisulfite