Amplification reporter with base-pairing oligomers
System, including methods, apparatus, and compositions, for performing amplification assays with an amplification reporter including a first oligomer and a second oligomer capable of base-pairing with one another below a melting temperature of the reporter. The reporter may have a detectable photoluminescence that is affected, such as reduced, by base-pairing of the first and second oligomers with one another. A target, such as a nucleic acid target sequence, may be amplified in at least one volume, such as a plurality of partitions, above the melting temperature, and photoluminescence of the reporter may be detected from the at least one volume below the melting temperature. A property of the target, such as a concentration of the target, may be determined based on the photoluminescence detected.
1. A method of analysis, the method comprising:
forming a mixture including a target;
forming a plurality of fluid volumes each containing a portion of the mixture, a reporter, and a pair of primers, wherein only a subset of the plurality of fluid volumes contain at least one copy of the target, the reporter including a first oligomer having a photoluminophore and a second oligomer having a quencher for the photoluminophore, the first oligomer and the second oligomer being capable of base-pairing with one another below the melting temperature of the reporter in the plurality of fluid volumes to quench photoluminescence of the photoluminophore, wherein the first oligomer and the second oligomer are not covalently linked to one another;
amplifying the target in the plurality of fluid volumes to produce an amplicon by extending the pair of primers at a temperature above the melting temperature of the reporter, wherein amplifying causes cleavage of the second oligomer, and wherein the cleavage increases the photoluminescence detectable from the photoluminophore of the first oligomer;
detecting photoluminescence from the photoluminophore contained by each fluid volume of the plurality of fluid volumes while the fluid volume is at a temperature below the melting temperature of the reporter, wherein fluid volumes of the plurality of fluid volumes that are positive for the target have more photoluminescence from the photoluminophore than fluid volumes of the plurality of fluid volumes that are negative for the target; and
determining a level of the target based on the photoluminescence detected.
2. The method of claim 1 , wherein the mixture includes the reporter.
3. The method of claim 1 , wherein the mixture includes the pair of primers.
4. The method of claim 1 , wherein the plurality of fluid volumes are droplets, and wherein the droplets are enclosed by an immiscible liquid to create an emulsion.
5. The method of claim 1 , wherein determining a level includes determining a number of the plurality of fluid volumes that contain the target or that do not contain the target based on the photoluminescence detected.
6. The method of claim 5 , wherein the level is a concentration of the target.
7. The method of claim 1 , wherein the second oligomer forms a hybrid with the target having a higher melting temperature than the melting temperature of the reporter.
8. The method of claim 1 , wherein amplifying includes exposing the plurality of fluid volumes to a plurality of thermal cycles, and the plurality of fluid volumes are maintained continuously above the melting temperature of the reporter throughout each thermal cycle of the plurality of thermal cycles.
9. The method of claim 1 , wherein each of the first and second oligomers has a chain of nucleotides, and wherein the chain of nucleotides of the second oligomer is longer than the chain of nucleotides of the first oligomer.