Patent ID: 7604937
Filing Date: 2009-10-20
Classification: C12Q

Abstract:
1. A method for comparing the amount of a target polynucleotide sequence between two samples comprising; providing a first reaction vessel one and a first reaction vessel two; wherein the first reaction vessel one comprises a first sample, a forward primer, and a reverse primer, wherein the forward primer comprises a target specific portion and a forward addressable primer portion, wherein the reverse primer comprises a target specific portion and a reverse addressable primer portion, wherein the forward primer or the reverse primer further comprises a first identifying portion; wherein the first reaction vessel two comprises a second sample, a forward primer and a reverse primer, wherein the forward primer comprises a target specific portion and a forward addressable primer portion, wherein the reverse primer comprises a target specific portion and a reverse addressable primer portion, wherein the forward primer or the reverse primer further comprises a second identifying portion; wherein the forward addressable primer portion in vessel one is different from the forward addressable primer portion in vessel two; performing a first encoding PCR in the first reaction vessel one, thereby forming a first encoding PCR product mixture comprising a first encoding PCR product, wherein the first encoding PCR product comprises the forward addressable primer portion, the first identifying portion, the target specific portions, and the reverse addressable primer portion, wherein the identity of target polynucleotide sequence is encoded with the forward addressable primer portion and the reverse addressable primer portion, and wherein the identity of the sample in the first encoding PCR is encoded by the first identifying portion; performing a second encoding PCR in the first reaction vessel two, thereby forming a second encoding PCR product mixture comprising a second encoding PCR product, wherein the second encoding PCR product comprises the forward addressable primer portion, the second identifying portion, the target specific portions, and the reverse addressable primer portion, wherein the identity of the target polynucleotide sequence is encoded with the forward addressable primer portion and the reverse addressable primer portion, and wherein the identity of the sample in the second encoding PCR is encoded by the second identifying portion; combining the first encoding PCR product mixture from the first reaction vessel one with the second encoding PCR product mixture from the first reaction vessel two to form a combined encoding PCR product mixture; providing a second reaction vessel, wherein the second reaction vessel comprises a forward address primer, a reverse address primer, a first labeling probe, and a second labeling probe, wherein the first labeling probe one is complementary to, or complementary to the complement of, the first identifying portion of the encoding PCR product from the first reaction vessel one, and wherein the second labeling probe one is complementary to, or complementary to the complement of, the second identifying portion of the encoding PCR product from the first reaction vessel two; adding an aliquot of the combined encoding PCR product mixture to the second reaction vessel, thereby forming a decoding amplification reaction in the second reaction vessel; performing a decoding amplification reaction in the second reaction vessel, wherein the forward address primer hybridizes to the complement of the forward addressable primer portion introduced into the encoding PCR product during the first encoding PCR and the second encoding PCR, wherein the reverse address primer hybridizes to the reverse addressable primer portion introduced in the encoding PCR product during the first encoding PCR and the second encoding PCR, wherein amplification of the encoding PCR product results in signal from the first labeling probe when the encoding PCR product is derived from the first encoding PCR, wherein amplification of the encoding PCR product results in signal from the second labeling probe when the encoding PCR product is derived from the second encoding PCR; detecting and quantifying the target polynucleotide in the two samples based on the presence and quantity of signal from the first labeling probe and the second labeling probe in the decoding reaction, wherein the decoding reaction is a real-time PCR; and, comparing the amount of the target polynucleotide sequence between two samples.