Compositions and methods for molecular labeling

Abstract

The invention provides barcode libraries and methods of making and using them including obtaining a plurality of nucleic acid constructs in which each construct comprises a unique N-mer and a functional N-mer and segregating the constructs into a fluid compartments such that each compartment contains one or more copies of a unique construct. The invention further provides methods for digital PCR and for use of barcode libraries in digital PCR.

1 - 31 . (canceled) 
     
     
         32 . A method for detecting a target nucleic acid using intensity multiplexing, the method comprising:
 partitioning a fluid containing target nucleic acid into one or more compartments with PCR reagents and a plurality of fluorescent probes that bind to a respective plurality of target sequences, wherein a plurality of the compartments include one template molecule;   amplifying nucleic acid in said compartments;   measuring fluorescent intensity from the compartments; and   identifying one of the target sequences based on the fluorescent intensity.   
     
     
         33 . The method of  claim 32 , wherein all of the fluorescent probes have the same color fluorophore. 
     
     
         34 . The method of  claim 32 , wherein there are at least two distinct target sequences and the amplifying step produces a distinct fluorescent intensity for each of the least two distinct target sequences. 
     
     
         35 . The method of  claim 34 , wherein the target sequences include one or more of EGFR, BRAF, KRAS, TERT, RNaseP, E1a, SMN1 and SMN2. 
     
     
         36 . The method of  claim 32 , wherein the fluid is partitioned with the target nucleic acid at a terminal or limiting dilution such that each compartment has zero, one or two template molecules. 
     
     
         37 . The method of  claim 32 , wherein the different fluorescent probes bind to the different respective target sequences with different binding affinities. 
     
     
         38 . The method of  claim 37 , wherein the different binding affinities are due to different number of matching or mis-matched bases between the probes and the target sequences. 
     
     
         39 . The method of  claim 32 , wherein the different fluorescent probes yield different fluorescent intensities due to different probe and/or primer concentrations. 
     
     
         40 . The method of  claim 32 , wherein the different fluorescent probes yield different fluorescent intensities due to (i) a portion of the probes lacking fluorophores; and/or (ii) the inclusion of one or more competitive inhibitors of probe binding. 
     
     
         41 . The method of  claim 32 , wherein the different fluorescent probes yield at least 2 different fluorescent intensities. 
     
     
         42 . A method for detecting target sequences, the method comprising:
 partitioning a fluid containing target nucleic acid into one or more compartments with PCR reagents and a plurality of fluorescent probes that bind to target sequences, wherein a plurality of the compartments include one template molecule;   amplifying nucleic acid in the compartments;   measuring fluorescent colors and intensities from the compartments; and   identifying one of the target sequences based on a combination of the measured colors and intensities.   
     
     
         43 . The method of  claim 42 , wherein each of the target sequences interacts with multiple probes to generate a unique signature of the colors and intensities. 
     
     
         44 . The method of  claim 42 , further comprising plotting the measured fluorescent colors and intensities from each compartment as points on a plot and identifying clusters of the points in different areas of the plot to identify the presence of the target sequences in the fluid. 
     
     
         45 . The method of  claim 42 , wherein the fluorescent probes each have one of two fluorophores. 
     
     
         46 . The method of claim  14 , wherein two fluorophores are measured in two channels and the method includes identifying greater than two distinct target sequences. 
     
     
         47 . The method of  claim 42 , wherein the different fluorescent probes bind to the different respective target sequences with different binding affinities. 
     
     
         48 . The method of  claim 42 , wherein the different fluorescent probes yield different fluorescent intensities due to different probe and/or primer concentrations. 
     
     
         49 . The method of  claim 42 , wherein the different fluorescent probes yield different fluorescent intensities due to (i) a portion of the probes lacking fluorophores; and/or (ii) the inclusion of one or more competitive inhibitors of probe binding. 
     
     
         50 . The method of  claim 42 , further comprising generating a 2D plot of fluorescence and associating distinct clusters in the 2D plot with different ones of the target sequences. 
     
     
         51 . The method of  claim 42 , wherein at least a first target sequence is bound only by probes with a first fluorophore, at least a second target sequence is bound only by probes with a second fluorophore, and at least a third target sequence is bound by a mixture of probes that include the first and second fluorophores. 
     
     
         52 . The method of  claim 42 , wherein the compartments are microfluidic droplets. 
     
     
         53 . The method of  claim 52 , wherein the droplets are in an emulsion. 
     
     
         54 . The method of  claim 42 , wherein the compartments are wells in a multiwell plate. 
     
     
         55 . The method of  claim 54 , wherein the multiwell plate has 5000 or more nanowells. 
     
     
         56 . The method of  claim 42 , wherein wells are not in fluid communication during amplification.