Encoding and decoding reactions for determining target molecules
View Patent ↗The present invention is directed to methods, reagents, and kits for detecting the presence or absence of (or quantifying) target polynucleotide sequences and proteins in at least one sample using encoding and decoding reactions. When a particular target polynucleotide is present in a sample for example, a reaction product is formed in the encoding reaction that includes addressable primer portions. At least one labeling probe and at least one address primer can be employed in the decoding amplification reaction thereby providing a detectable signal value depending upon whether a sequence is present or absent. In some embodiments, the encoding comprises a ligation reaction with linker probes, and single nucleotide polymorphisms (SNPs) are analyzed.
1. A kit for comparing the amount of a target polynucleotide sequence between a first sample and a second sample comprising;
A) a first reaction vessel one comprising;
a forward primer, wherein the forward primer comprises a target specific portion and an addressable forward primer portion;
a reverse primer, wherein the reverse primer comprises a target specific portion and an addressable reverse primer portion, wherein the forward primer, the reverse primer, or both, comprise a first identifying portion;
B) a first reaction vessel two comprising;
a forward primer, wherein the forward primer comprises a target specific portion and an addressable forward primer portion;
a reverse primer, wherein the reverse primer comprises a target specific portion and an addressable reverse primer portion, wherein the forward primer, the reverse primer, or both, comprise a second identifying 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 first sample is encoded by the first identifying portion and the identity of the second sample is encoded by the second identifying portion;
C) a second reaction vessel comprising;
a forward address primer and a reverse address primer;
wherein the forward addressable primer portion in vessel one is different from the forward addressable primer portion in vessel two; and,
D) a PCR master mix comprising;
a polymerase, dNTPs, a buffer, a first labeling probe and a second labeling probe, wherein the first labeling probe is complementary to, or complementary to the complement of, the first identifying portion, and wherein the second labeling probe is complementary to, or complementary to the complement of, the second identifying portion.
2. The kit according to claim 1 wherein the first labeling probe, the second labeling probe, or both, comprise PNA.
3. The kit according to claim 1 wherein the first labeling probe, the second labeling probe, or both, are a 5′-nuclease cleavable probe.
4. A kit for comparing the amount of a target polynucleotide sequence between two samples comprising;
A) a first reaction vessel one comprising;
a first ligation probe, wherein the first ligation probe comprises a target specific portion and an addressable forward primer portion;
a second ligation probe, wherein the second ligation probe comprises a target specific portion and an addressable reverse primer portion, wherein the first ligation probe, the second ligation probe, or both, comprise a first identifying portion;
B) a first reaction vessel two comprising;
a first ligation probe, wherein the first ligation probe comprises a target specific portion and an addressable forward primer portion;
a second ligation probe, wherein the second ligation probe comprises a target specific portion and an addressable reverse primer portion, wherein the first ligation probe, the second ligation probe, or both, comprise a second identifying 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 first sample is encoded by the first identifying portion and wherein the identity of the second sample is encoded by the second identifying portion;
C) a second reaction vessel comprising;
a forward address primer and a reverse address primer;
wherein the forward addressable primer portion in vessel one is different from the forward addressable primer portion in vessel two;
D) a PCR master mix, wherein the PCR master mix comprises; a polymerase, dNTPs, a buffer, a first labeling probe, and a second labeling probe, wherein the first labeling probe is complementary to, or complementary to the complement of, the first identifying portion, and wherein the second labeling probe is complementary to, or complementary to the complement of, the second identifying portion.
5. The kit according to claim 4 further comprising;
E) a ligase master mix, wherein the ligase master mix comprises a ligase and a buffer.
6. The kit according to claim 4 wherein the first labeling probe, the second labeling probe, or both the first labeling probe and the second labeling probe comprise PNA.
7. The kit according to claim 4 wherein the first labeling probe, the second labeling probe, or both, are a 5′-nuclease cleavable probe.