| 摘要 |
A real-time method of detecting the presence and/or amount of a methylated or unmethylated gene of interest in a DNA- containing sample, comprises the steps of: (a) contacting the DNA-containing sample with a reagent which selectively modifies unmethylated cytosine residues in the DNA to produce detectable modified residues but which does not modify methylated cytosine residues (b) amplifying at least a portion of the methylated or unmethylated gene of interest using at least one primer pair, at least one primer of which is designed to bind only to the sequence of methylated or unmethylated DNA following treatment with the reagent, wherein at least one primer in the primer pair is a primer containing a stem loop structure carrying a donor and an acceptor moiety of a molecular energy transfer pair arranged such that in the absence of amplification, the acceptor moiety quenches fluorescence emitted by the donor moiety upon excitation and during amplification, the stem loop structure is disrupted so as to separate the donor and acceptor moieties sufficiently to produce a detectable fluorescence signal which is detected in real-time to provide an indication of the gene copy number of the methylated or unmethylated gene of interest (c) quantifying the results of the real-time detection against a standard curve for the methylated or unmethylated gene of interest to produce an output of gene copy number. The method may be characterised in that the amplification is considered valid where the cycle threshold value is less than (40). Alternatively the method, which may be carried out in real-time or at end point, is characterised in that the portion of the gene which is amplified is between approximately (50) and 250bp, especially for plasma and serum samples. |
