Multiplexed digital assays with combinatorial use of signals

Abstract

System, including methods, apparatus, and compositions, for performing a multiplexed digital assay on a greater number of targets through combinatorial use of signals.

We claim: 
     
         1 . A method of determining an amount of target linkage, the method comprising:
 partitioning an aqueous phase containing a sample into a plurality of partitions, the sample including nucleic acid providing a first target and a second target;   amplifying the first target and the second target in partitions;   collecting amplification data from partitions;   determining a number of partitions containing the first target and not the second target, a number of partitions containing the second target and not the first target, a number of partitions containing both targets, and a number of partitions containing neither target; and   calculating a value indicating a percent linkage of the first target and the second target to one another in the sample using a plurality of the numbers.   
     
     
         2 . The method of  claim 1 , wherein the value indicates a percent linkage greater than 0% and less than 100%. 
     
     
         3 . The method of  claim 2 , wherein the step of partitioning is performed with nucleic acid isolated from a source in which the first and second targets are substantially always linked to one another, such that a difference between 100% linkage and the percent linkage represents a percent fragmentation of the nucleic acid between the first target and the second target. 
     
     
         4 . The method of  claim 1 , wherein the step of amplifying includes a step of thermally cycling partitions and a step of performing a polymerase chain reaction in partitions. 
     
     
         5 . The method of  claim 1 , wherein the step of partitioning includes a step of forming droplets of an emulsion. 
     
     
         6 . The method of  claim 1 , wherein each partition include a first probe to detect amplification of the first target and a second probe to detect amplification of the second target, and wherein the first probe and the second probe are different from one another. 
     
     
         7 . The method of  claim 6 , wherein each probe includes an oligonucleotide and a fluorophore. 
     
     
         8 . The method of  claim 7 , wherein each probe includes a quencher for the fluorophore of the probe. 
     
     
         9 . The method of  claim 1 , wherein the step of collecting amplification data includes a step of detecting an intensity of light emitted by at least one fluorophore from partitions. 
     
     
         10 . The method of  claim 9 , wherein the step of determining includes a step of comparing (i) the intensity of light emitted by a fluorophore to (ii) a threshold. 
     
     
         11 . A composition for performing a multiplexed digital amplification assay, comprising:
 an emulsion including droplets, each droplet including a portion of a same aqueous phase;   wherein the aqueous phase includes three probes and is configured to amplify a first target, a second target, and a third target,   wherein each of the three probes is configured to detect amplification of a different one of the targets,   wherein the three probes are labeled with a total of no more than two different fluorophores, and   wherein each droplet of a subset of the droplets contains the first target and the second target but not the third target, each droplet of another subset of the droplets contains the first target and the third target but not the second target, each droplet of yet another subset of the droplets contains the second target and the third target but not the first target, and each droplet of still another subset of the droplets contains none of the targets.   
     
     
         12 . The composition of  claim 11 , wherein the emulsion includes a continuous phase surrounding each droplet, and wherein the continuous phase includes an oil. 
     
     
         13 . The composition of  claim 11 , wherein the aqueous phase includes a sample that provides the first, second, and third targets. 
     
     
         14 . The composition of  claim 11 , wherein each droplet of a subset of the droplets contains only the first target of the three targets, each droplet of a subset of the droplets contains only the second target of the three targets, and each droplet of a subset of the droplets contains only the third target of the three targets. 
     
     
         15 . The composition of  claim 11 , wherein each droplet of a subset of the droplets contains each of the first, second, and third targets. 
     
     
         16 . The composition of  claim 11 , wherein each probe includes an oligonucleotide attached to a fluorophore. 
     
     
         17 . The composition of  claim 11 , wherein each droplet includes a heat-stable polymerase. 
     
     
         18 . The composition of  claim 11 , wherein the aqueous phase is configured for PCR amplification of each target, and wherein each droplet includes primers for amplification of each of the three targets. 
     
     
         19 . The composition of  claim 11 , wherein each probe includes a quencher and one of the fluorophores. 
     
     
         20 . A method of quantifying targets with the composition of  claim 11 , the method comprising:
 detecting light emitted by each of the fluorophores from a plurality of the droplets of the composition of  claim 11 ; and   determining a number of droplets that are positive or a number of droplets that are negative for each of the targets.