A method for identifying in a sample, one or more target ribonucleic acid molecules differing in sequence from other ribonucleic acid molecules in the sample due to alternative splicing, alternative transcript, alternative start site, alternative coding sequence, alternative non-coding sequence, exon insertion, exon deletion, intron insertion, translocation, mutation, or other rearrangement at the genome level, said method comprising:
- providing a sample containing one or more target ribonucleic acid molecules potentially differing in sequence from other ribonucleic acid molecules;
- contacting the sample with one or more enzymes capable of digesting dU containing nucleic acid molecules potentially present in the sample;
- providing one or more oligonucleotide primers, each primer being complementary to the one or more target ribonucleic acid molecules;
- blending the contacted sample, the one or more oligonucleotide primers, a deoxynucleotide mix including dUTP, and a reverse-transcriptase to form a reverse-transcription mixture;
- generating complementary deoxyribonucleic acid (cDNA) molecules in the reverse transcription mixture, each cDNA molecule comprising a nucleotide sequence that is complementary to the target ribonucleic acid molecule and contains dU;
- providing one or more oligonucleotide primer sets, each primer set comprising (a) a first oligonucleotide primer comprising a nucleotide sequence that is complementary to a portion of a cDNA nucleotide sequence adjacent to the target ribonucleic acid molecule sequence complement of the cDNA, and (b) a second oligonucleotide primer comprising a nucleotide sequence that is complementary to a portion of an extension product formed from the first oligonucleotide primer;
- blending the reverse transcription mixture containing the cDNA molecules, the one or more oligonucleotide primer sets, a deoxynucleotide mix including dUTP, and a polymerase to form a polymerase reaction mixture;
- subjecting the polymerase chain reaction mixture to one or more polymerase chain reaction cycles comprising a denaturation treatment, a hybridization treatment, and an extension treatment thereby forming one or more different primary extension products;
- providing one or more oligonucleotide probe sets, each probe set comprising (a) a first oligonucleotide probe having a 5′ primer-specific portion and a 3′ target sequence-specific portion, and (b) a second oligonucleotide probe having a 5′ target sequence-specific portion and a 3′ primer-specific portion, wherein the first and second oligonucleotide probes of a probe set are configured to hybridize, in a base specific manner, on complementary portions of a primary extension product corresponding to the target ribonucleic acid molecule sequence;
- contacting the primary extension products with a ligase and the one or more oligonucleotide probe sets to form a ligation reaction mixture;
- subjecting the ligation reaction mixture to one or more ligation reaction cycles whereby the first and second probes of the one or more oligonucleotide probe sets are ligated together to form ligated product sequences in the ligase reaction mixture, wherein each ligated product sequence comprises the 5′ primer-specific portion, the target-specific portions, and the 3′ primer-specific portion;
- providing one or more secondary oligonucleotide primer sets, each secondary oligonucleotide primer set comprising (a) a first secondary oligonucleotide primer comprising the same nucleotide sequence as the 5′ primer-specific portion of the ligated product sequence and (b) a second secondary oligonucleotide primer comprising a nucleotide sequence that is complementary to the 3′ primer-specific portion of the ligated product sequence;
- blending the ligated product sequences, the one or more secondary oligonucleotide primer sets with one or more enzymes capable of digesting deoxyuracil (dU) containing nucleic acid molecules, a deoxynucleotide mix including dUTP, and a DNA polymerase to form a second polymerase chain reaction mixture;
- subjecting the second polymerase chain reaction mixture to conditions suitable for digesting deoxyuracil (dU) containing nucleic acid molecules present in the second polymerase chain reaction mixture, and one or more polymerase chain reaction cycles comprising a denaturation treatment, a hybridization treatment, and an extension treatment thereby forming secondary extension products;
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- detecting and distinguishing the secondary extension products in the sample thereby identifying the presence of one or more ribonucleic acid molecules differing in sequence from other ribonucleic acid molecules in the sample due to alternative splicing, alternative transcript, alternative start site, alternative coding sequence, alternative non-coding sequence, exon insertion, exon deletion, intron insertion, translocation, mutation, or other rearrangement at the genome level.