Multiplexed digital assay

Abstract

Method of performing a multiplexed digital assay. The method may include (1) partitioning a sample containing at least two distinct targets into droplets; (2) amplifying the at least two distinct targets within the droplets; (3) detecting light from the droplets indicative of the presence or absence of each distinct target in each droplet; and (4) calculating a level of each distinct target based on the detected light.

We claim: 
     
         1 . A method of performing a multiplexed digital assay, comprising:
 partitioning a sample containing at least two distinct targets into droplets;   amplifying the at least two distinct targets within the droplets;   detecting light from the droplets indicative of the presence or absence of each distinct target in each droplet; and   calculating a level of each distinct target based on the detected light.   
     
     
         2 . The method of  claim 1 , wherein each of the distinct targets is a nucleic acid. 
     
     
         3 . The method of  claim 1 , wherein each droplet contains only about one target. 
     
     
         4 . The method of  claim 1 , wherein the droplets have an average diameter of less than about 100 micrometers. 
     
     
         5 . The method of  claim 1 , wherein each droplet further includes target-specific reagents. 
     
     
         6 . The method of  claim 1 , wherein the light detected for each distinct target is distinguishable. 
     
     
         7 . The method of  claim 1 , wherein a distinct reporter dye reports amplification of each distinct target, and wherein the distinct reporter dyes differ in emission spectra so that they may be distinguished. 
     
     
         8 . The method of  claim 7 , wherein the reporter dyes are selected from the group consisting of fluorescein, fluorescein derivatives, and rhodamine dyes. 
     
     
         9 . The method of  claim 1 , wherein the level is a concentration in the sample. 
     
     
         10 . The method of  claim 1 , wherein the step of calculating a level includes a step of determining a total number of droplets containing each distinct target and a step of determining the total number of all droplets. 
     
     
         11 . The method of  claim 1 , wherein a first reporter signals the presence or absence of a first group of targets and a second reporter signals the presence or absence of a second group of targets. 
     
     
         12 . The method of  claim 11 , wherein the first group of targets is from a first chromosome and the second group of targets is from a second chromosome. 
     
     
         13 . The method of  claim 12 , wherein the first chromosome is chromosome 18 and the second chromosome is chromosome 21. 
     
     
         14 . The method of  claim 12 , further comprising counting the number present of the first or second chromosome. 
     
     
         15 . The method of  claim 1 , wherein the level is a copy number. 
     
     
         16 . The method of  claim 1 , wherein the two distinct targets differ by a single nucleotide. 
     
     
         17 . The method of  claim 1 , wherein the step of detecting light is performed with the droplets disposed in a flow stream. 
     
     
         18 . The method of  claim 1 , wherein the step of detecting light is performed at different temperatures. 
     
     
         19 . The method of  claim 1 , further comprising a step of producing one or more melting curves for each droplet. 
     
     
         20 . The method of  claim 1 , wherein a reporter dye reports amplification of each distinct target, and wherein the number of distinct targets exceeds the number of distinct reporter dyes. 
     
     
         21 . The method of  claim 1 , wherein the droplets contain an intercalating dye that reports amplification of each of the distinct targets.