Methods for quantitating nucleic acids using coupled ligation and amplification

A method for quantitating at least one target nucleic acid sequence in a sample comprising:

• combining at least one target nucleic acid sequence with a probe set for each target nucleic acid sequence, the probe set comprising (a) at least one first probe, comprising a first target-specific portion, and (b) at least one second probe, comprising a second target-specific portion and a 3′ primer-specific portion, wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on the at least one target nucleic acid sequence, and wherein at least one probe in each probe set further comprises a promoter or its complement, and wherein at least one probe in each probe set further comprises at least one addressable support-specific portion, and when the at least one first probe comprises the at least one addressable support-specific portion, the at least one first probe further comprises a 5′ primer-specific portion, and wherein the at least one addressable support-specific portion is located between the primer-specific portion and the target-specific portion of the at least one probe in each probe set;
• to form a ligation reaction mixture;
• subjecting the ligation reaction mixture to at least one cycle of ligation, wherein adjacently hybridized probes are ligated to form a ligation product comprising the first and second target specific portions, the at least one addressable support-specific portion, the 3′ primer-specific portion, and the promoter sequence or its complement;
• combining the ligation product with at least one primer set comprising at least one second primer comprising a sequence complementary to the 3′ primer-specific portion of the ligation product and a DNA polymerase, to form a first amplification reaction mixture;
• subjecting the first amplification reaction mixture to at least one cycle of amplification to generate a first amplification product comprising the promoter sequence;
• combining the first amplification product with an RNA polymerase and a ribonucleoside triphosphate solution comprising at least one of rATP, rCTP, rGTP, or rUTP, to form a transcription reaction mixture;
• incubating the transcription reaction mixture under appropriate conditions to generate an RNA transcription product;
• detecting the RNA transcription product or a portion of the RNA transcription product using the at least one addressable support-specific portion;
• and
• quantitating the at least one target nucleic acid sequence.

A method for quantitating at least one target nucleic acid sequence in a sample comprising:

• combining at least one target nucleic acid sequence with a probe set for each target nucleic acid sequence, the probe set comprising (a) a first probe, comprising a first target-specific portion and a 5′ primer-specific portion, and (b) a second probe, comprising a second target-specific portion and a 3′ primer-specific portion, wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on the at least one target nucleic acid sequence, and wherein at least one probe in each probe set further comprises at least one addressable support-specific portion located between the primer-specific portion and the target-specific portion of the at least one probe in each probe set;
• to form a ligation reaction mixture;
• subjecting the ligation reaction mixture to at least one cycle of ligation, wherein adjacently hybridized probes are ligated to one another to form a ligation product comprising the 5′ primer-specific portion, the first and second target specific portions, the at least one addressable support-specific portion, and the 3′ primer-specific portion;
• combining the ligation product with:
• (a) at least one primer set comprising:
• (i) at least one first primer comprising the sequence of the 5′ primer-specific portion of the ligation product, and (ii) at least one second primer comprising a sequence complementary to the 3′ primer-specific portion of the ligation product;
• and (b) a DNA polymerase;
• to form a first amplification reaction mixture;
• subjecting the first amplification reaction mixture to at least one cycle of amplification to generate a first amplification product;
• combining the first amplification product with either at least one first primer, or at least one second primer for each primer set, but not both first and second primers, to form a second amplification reaction mixture;
• subjecting the second amplification reaction mixture to at least one cycle of amplification to generate a second amplification product;
• detecting the second amplification product or a portion of the second amplification product using the at least one addressable support-specific portion;
• and
• quantitating the expression of the at least one target nucleic acid sequence.