Method for relative quantification of nucleic acid sequence, expression, or copy changes, using combined nuclease, ligation, and polymerase reactions

A method for identifying a presence of one or more target nucleotide sequences in a sample comprising:

• providing a sample potentially containing the one or more target nucleotide sequences;
• providing one or more oligonucleotide probe sets, each set comprising (a) a first oligonucleotide probe having a 5′ primer-specific portion and a target-specific portion, and (b) a second oligonucleotide probe having a 3′ primer-specific portion, a target specific portion, and a 5′ nucleotide sequence, wherein said 5′ nucleotide sequence is complementary to at least a portion of the 3′ primer-specific portion and hybridizes to said complementary portion of the 3′ primer-specific portion to form a hairpinned second oligonucleotide probe when said second probe is not hybridized to a target nucleotide sequence, wherein the first and second oligonucleotide probes of a probe set are configured to hybridize adjacent to one another on the target nucleotide sequence with a junction between the first and second oligonucleotide probes, and wherein, in a probe set, the target specific portion of the second oligonucleotide probe has an overlapping identical nucleotide at the junction with the first oligonucleotide probe;
• contacting the sample and the one or more oligonucleotide probe sets under conditions effective for first and second oligonucleotide probes of a probe set to hybridize at adjacent positions in a base specific manner to their corresponding target nucleotide sequences, if present in the sample, wherein upon hybridization the overlapping identical nucleotide of the second oligonucleotide probe forms a flap at the junction comprising the overlapping identical nucleotide;
• cleaving the overlapping identical nucleotide of the second oligonucleotide probe with an enzyme having 5′ nuclease activity, thereby liberating a phosphate at the second oligonucleotide probe's 5′ end;
• ligating first and second oligonucleotide probes of the one or more oligonucleotide probe sets together at the junction to form ligated product sequences, wherein each ligated product sequence comprises the 5′ primer-specific portion, the target-specific portions, and the 3′ primer-specific portions of the first and second oligonucleotide probes of an oligonucleotide probe set;
• detecting the ligated product sequences in the sample;
• and
• identifying the presence of one or more target nucleotide sequences in the sample based on said detecting.