Signal encoding and decoding in multiplexed biochemical assays
This disclosure provides methods, systems, compositions, and kits for the multiplexed detection of a plurality of analytes in a sample. In some examples, this disclosure provides methods, systems, compositions, and kits wherein multiple analytes may be detected in a single sample volume by acquiring a cumulative measurement or measurements of at least one quantifiable component of a signal. In some cases, additional components of a signal, or additional signals (or components thereof) are also quantified. Each signal or component of a signal may be used to construct a coding scheme which can then be used to determine the presence or absence of any analyte.
1. A method of detecting the presence or absence of at least seven polynucleotide analytes, in any combination of presence or absence, in a single sample solution volume, the method comprising:
a) providing a sample solution volume comprising or potentially comprising, at least one of at least seven polynucleotide analytes, and comprising at least seven hybridization probes, each hybridization probe corresponding to one of the at least seven polynucleotide analytes, wherein each hybridization probe comprises at least one fluorophore selected from four fluorophores, and wherein each hybridization probe, when excited and when contacted with its corresponding polynucleotide analyte, generates a cumulative intensity signal, further wherein each generated cumulative intensity signal comprises at least one signal generated by excitement of at least one of the four fluorophores;
b) contacting said sample solution volume with the plurality of hybridization probes and exciting the hybridization probes to generate the cumulative intensity signal(s) if one or more of the at least seven polynucleotide analytes is present in the sample volume; and
c) measuring the cumulative intensity signal(s), thereby generating a cumulative signal measurement, wherein the cumulative signal measurement non-degenerately indicates the presence or absence of the at least seven polynucleotide analytes, in any combination of presence or absence,
wherein said method non-degenerately detects the presence or absence of the at least seven polynucleotide analytes in said single sample solution volume, in any combination of presence or absence, without requiring any step of immobilization of said polynucleotide analytes, physical separation of said polynucleotide analytes, or mass spectrometry.
2. The method of claim 1 , wherein said hybridization probes comprise an identical fluorophore, and wherein said plurality of hybridization probes are specific for different polynucleotide analytes.
3. The method of claim 1 , wherein said detecting is performed with a plurality of hybridization probes comprising different fluorophores, wherein said plurality of hybridization probes are specific for different analytes.
4. The method of claim 1 , wherein said cumulative intensity signal(s) are generated during a polymerase chain reaction.
5. The method of claim 1 , further comprising transmitting information concerning the presence or absence of said polynucleotide analytes through a computer network.
6. The method of claim 1 , further comprising providing information concerning the presence or absence of said polynucleotide analytes to a health care professional, wherein the health care professional makes a clinical decision based on said information.
7. The method of claim 1 , wherein at least one step of said method is performed using a computer.
8. The method of claim 7 , wherein said computer is located on a remote server.
9. The method of claim 7 , wherein said computer is located on a thermal cycler.