Abstract: An improved multiplex ligation-dependent amplification method is disclosed for detecting the presence of specific methylated sites in a single stranded target nucleic acid, while simultaneously, the quantification of the target nucleic acid sequence can be performed, using a plurality of probe sets of at least two probes, each of which includes a target specific region and non-complementary region containing a primer binding site. At least one of the probes further includes the sequence of one of the strands of a double stranded recognition site of a methylation sensitive restriction enzyme. The probes belonging to the same set are ligated together when hybridised to the target nucleic acid sequence, the hybrid is subjected to digestion by the methylation sensitive restriction enzyme, resulting in non-methylated recognition sites being cleaved. The probes of the uncleaved (methylated) hybrid are subsequently amplified by a suitable primer set.

Abstract: Described is an improved multiplex ligation-dependent amplification method for detecting the presence and quantification of at least one specific single stranded target nucleic acid sequence in a sample using a plurality of probe sets of at least two probes, each of which includes a target specific region and a non-complementary region comprising a primer binding site. The probes belonging to the same set are ligated together when hybridised to the target nucleic acid sequence and amplified by a suitable primer set. By using a femtomolar amount of the probes a large number of different probe sets can be used to simultaneously detect and quantify a corresponding large number of target sequences with high specificity.