Methods and devices for digital pcr

Abstract

A method comprises flowing a plurality of sample droplets in a continuous flow of a carrier fluid, immiscible with the sample droplets, such that the droplets are separated from each other by the carrier fluid, wherein an average number of copies of target nucleic acid contained in each droplet the plurality of sample droplets is one or fewer. The method may further comprise subjecting the droplets to thermal cycling sufficient to allow amplification of the target nucleic acid, and detecting one or more of the presence or absence of amplified target nucleic acid in the droplets.

1 . (canceled) 
     
     
         2 . A method of quantifying a target nucleic acid of a sample using a system comprising a sample preparation component, a thermal cycler component, and a detection component, the method comprising:
 in the sample preparation component:
 drawing sample from individual wells of a sample holder, the sample comprising nucleic acid and reagent for amplification of target nucleic acid; 
 forming a set of droplets by flowing an immiscible carrier fluid into contact with a flow of the sample drawn from the wells of a sample holder and thereby segmenting the sample into the set of droplets; 
 subjecting the set of droplets to an amplification reaction by thermal cycling the set of droplets in the thermal cycler component; 
   after the subjecting, flowing individual droplets of the set of droplets to a detection region of the detection component;   detecting as each individual droplet flows past the detection region for a presence or an absence of an amplification product of the target nucleic acid;   calculating a total number of the individual droplets detected to contain the amplification product; and   determining the starting number of copies of the target nucleic acid based on the calculated total number.   
     
     
         3 . The method of  claim 2 , wherein the amplification reaction comprises one or more of a polymerase chain reaction (PCR), a ligase chain reaction (LCR), a transcription based amplification system (TAS), a nucleic acid sequence-based amplification (NASBA), a strand displacement amplification (SDA), a rolling circle amplification (RCA), and a hyper-branched RCA (HRCA). 
     
     
         4 . The method of  claim 2 , wherein the amplification reaction is performed until a near-saturation concentration of the amplification product is produced in each droplet containing the amplification product. 
     
     
         5 . The method of  claim 2 , wherein the amplification reaction comprises thermal cycling the set of droplets through multiple thermal cycles. 
     
     
         6 . The method of  claim 5 , wherein the multiple thermal cycles ranges from 20 to 40 thermal cycles. 
     
     
         7 . The method of  claim 5 , wherein the detecting is performed at one or more of the thermal cycles. 
     
     
         8 . The method of  claim 5 , wherein the detecting occurs at each of the multiple thermal cycles. 
     
     
         9 . The method of  claim 2 , further comprising altering a concentration of the sample by a dilution factor, wherein determining the starting number of copies of the target nucleic acid comprises use of the dilution factor. 
     
     
         10 . The method of  claim 2 , wherein at least some droplets of the set of droplets do not contain any copy of the target nucleic acid. 
     
     
         11 . The method of  claim 10 , wherein from 50% to 90% of droplets in the set of droplets do not contain any copy of the target nucleic acid. 
     
     
         12 . The method of  claim 2 , wherein the target nucleic acid comprises DNA. 
     
     
         13 . The method of  claim 12 , wherein the DNA comprises genomic DNA. 
     
     
         14 . The method of  claim 2 , wherein the sample comprises cDNA. 
     
     
         15 . The method of  claim 14 , further comprising performing reverse transcription of mRNA of the sample to produce the cDNA. 
     
     
         16 . The method of  claim 2 , wherein the set of droplets comprises a reagent for the amplification reaction. 
     
     
         17 . The method of  claim 16 , wherein the reagent comprises one or more primers. 
     
     
         18 . The method of  claim 2 , wherein the sample contains at least 10 copies of the target nucleic acid and at least 10% of the sample is segmented. 
     
     
         19 . The method of  claim 2 , wherein a volume of each droplet of the set of droplets ranges from 0.1 μl to 500 nl. 
     
     
         20 . The method of  claim 2 , wherein the set of droplets comprises at least 5 droplets. 
     
     
         21 . The method of  claim 2 , wherein the detecting comprises detecting the amplification product in fewer than 50% of droplets in the set of droplets. 
     
     
         22 . The method of  claim 2 , wherein each droplet of the set of droplets are wrapped in and carried by a continuous flow of the immiscible carrier fluid during the amplification reaction and detecting.