Methods and kits for selectively amplifying, detecting or quantifying target DNA with specific end sequences

A method for detecting and/or quantifying a hypomethylated target DNA fragment in a sample containing a corresponding hypermethylated target DNA fragment, wherein both the hypomethylated target DNA fragment and the corresponding hypermethylated target DNA fragment contain a methylation-sensitive restriction enzyme recognition site, the method comprising:

• (a) digesting the sample with a methylation-sensitive restriction enzyme which recognizes the methylation-sensitive restriction enzyme recognition site;
• (b) contacting the digested sample with a stem-loop primer, wherein a 3′ portion of the stem-loop primer is complementary to the 3′ end of a strand of a digested DNA fragment at the methylation-sensitive restriction enzyme recognition site, and a 5′ portion of the stem-loop primer is capable of forming a stem-loop structure;
• (c) denaturing the mixture of step (b) to denature the digested DNA fragment and stem-loop primer;
• (d) annealing the stem-loop primer to the strand of the restriction enzyme-digested DNA fragment via the 3′ portion thereof and performing an extension reaction by using the strand of the digested DNA fragment as a template, wherein the stem-loop primer does not efficiently anneal to the hypermethylated target DNA fragment which is not cut or digested;
• and
• (e) detecting and/or quantifying the extension product with a PCR assay, wherein one primer of the primer pair used in the PCR assay is specific to the stem-loop primer,
• wherein results of the PCR assay indicate the presence and/or quantity of the hypomethylated target DNA fragment in the sample.