providing a sample potentially containing one or more target nucleic acid molecules;

subjecting the sample to a bisulfite treatment to convert, in the nucleic acid molecules of the sample, unmethylated cytosine residues, but not methylated cytosine residues, into uracil residues;

providing one or more primary oligonucleotide primer sets, each set characterized by (a) a first oligonucleotide primer, having a target-specific portion and a 5′ upstream secondary primer-specific portion, wherein the target-specific portion is suitable for hybridization on a first strand of the target nucleic acid molecule in which unmethylated cytosines have been converted to uracil, and (b) a second oligonucleotide primer, having a target-specific portion and a 5′ upstream secondary primer-specific portion, wherein the target-specific portion is suitable for hybridization on a polymerase extension product of the first strand or on a second strand of the target nucleic acid molecule, either of which having unmethylated cytosines converted to uracil and wherein the first and second oligonucleotide primers of each set contain the same 5′ upstream secondary primer-specific-portion;

providing a polymerase;

blending the sample, the primary oligonucleotide primer set, and the polymerase to form a primary polymerase chain reaction mixture;

subjecting the primary polymerase chain reaction mixture to two or more polymerase chain reaction cycles comprising a denaturation treatment, wherein hybridized nucleic acid sequences are separated, a hybridization treatment, wherein the target-specific portions of the primary oligonucleotide primer sets hybridize to the target nucleic acid molecules with unmethylated cytosines converted to uracil or to extension products of such modified target nucleic acid molecules, and an extension treatment, wherein the hybridized primary oligonucleotide primers are extended to form primary extension products complementary to the target nucleic acid molecules with unmethylated cytosines converted to uracil;

providing a secondary oligonucleotide primer set characterized by (a) a first secondary primer containing the 5′ upstream portion of the first oligonucleotide primer of the primary oligonucleotide primer set, and (b) a second secondary primer containing the 5′ upstream portion of the second oligonucleotide primer of the primary oligonucleotide primer set;

blending the primary extension products, the secondary oligonucleotide primer set, and the polymerase to form a secondary polymerase chain reaction mixture;

subjecting the secondary polymerase chain reaction mixture to two or more polymerase chain reaction cycles comprising a denaturation treatment, wherein hybridized nucleic acid sequences are separated, a hybridization treatment, wherein the secondary oligonucleotide primers hybridize to the primary extension products, and an extension treatment, wherein the hybridized secondary oligonucleotide primers are extended to form secondary extension products complementary to the primary extension products;

providing a plurality of oligonucleotide probe sets, each set characterized by (a) a first oligonucleotide probe, having a secondary extension product-specific portion and a detectable reporter label, and (b) a second oligonucleotide probe, having a secondary extension product-specific portion, wherein the oligonucleotide probes in a particular set are suitable for ligation together when hybridized on a complementary secondary extension product, but have a mismatch which interferes with such ligation when hybridized to any other nucleic acid molecule present in the sample;

providing a ligase;

blending the secondary extension products, the plurality of oligonucleotide probe sets, and the ligase to form a ligase detection reaction mixture;

subjecting the ligase detection reaction mixture to one or more ligase detection reaction cycles comprising a denaturation treatment, wherein any hybridized oligonucleotides are separated from the secondary extension product, and a hybridization treatment, wherein the oligonucleotide probe sets hybridize in a base-specific manner to their respective secondary extension products, if present, and ligate to one another to form a ligation product containing (a) the detectable reporter label and (b) the secondary extension product-specific portions connected together, wherein the oligonucleotide probe sets may hybridize to other nucleic acid molecules but do not ligate together due to a presence of one or more mismatches and individually separate during the denaturation treatment; and

detecting the reporter labels of the ligation products, thereby indicating the presence of two or more methylated cytosine bases in the target nucleotide sequences in the sample.