Quantitative methylation detection in DNA samples

Abstract

Described is a method for methylation detection in a DNA sample. An isolated genomic DNA sample is treated in a manner capable of distinguishing methylated from unmethylated cytosine bases. The pretreated DNA is amplified using at least one oligonucleotide primer, a polymerase and a set of nucleotides of which at least one is labeled with a first type of label. A sequence-specific oligonucleotide probe, marked with a second type of label, hybridizes to the amplification product and a FRET reaction occurs if a labeled oligonucleotide is present in close proximity in the amplification product. The method determines the level of methylation of a sample by measuring the extent of fluorescence resonance energy transfer (FRET) between the donor and acceptor fluorophore.

1 . A method for the cytosine methylation detection in a DNA sample, comprising the following steps: 
 a) a genomic DNA sample is treated in a manner capable of distinguishing methylated from unmethylated cytosine bases;    b) the pre-treated DNA is amplified using at least one oligonucleotide primer, a polymerase and a set of nucleotides of which at least one is marked with a first type of label;    c) a sequence-specific oligonucleotide or oligomer probe is hybridized to the amplification product and a fluorescence resonance energy transfer (FRET) occurs if the oligonucleotide or oligomer probe, marked with a second type of label, binds in close proximity to one of the labeled nucleotides that was incorporated into the amplification product;    d) the level of methylation of the sample is determined by the level of interaction between said first and second type of label.    
     
     
         2 . A composition of pre-treated genomic DNA according to  claim 1  for the determination of the methylation status of a corresponding genomic DNA.  
     
     
         3 . A diagnostic kit for the detection of the methylation of cytosine bases in genomic DNA samples according to  claim 1 , comprising reagents for the selective deamination of cytosine bases in genomic DNA, one or more primers and labeled nucleotides for the amplification step, a detectable probe and optionally protocols or instructions for the method of  claim 1.