Methods and systems for detecting biological components

Abstract

Methods for the detection of components from biological samples are provided. In certain aspects, the methods may be used to detect and/or quantify specific components in a biological sample, such as tumor cells (e.g., circulating tumor cells). Systems and devices for practicing the subject methods are also provided.

What is claimed is: 
     
         1 . A method of amplifying one or more target polynucleotides, the method comprising:
 (a) lysing one or more polynucleotide-containing components from a sample in a droplet to form a lysate in a lysate droplet, wherein the lysate comprises the one or more target polynucleotides and the lysate droplet is in an immiscible carrier fluid;   (b) adding to the lysate reagents for performing a nucleic acid amplification reaction to form an amplification mixture in an amplification droplet, wherein the amplification droplet is in the immiscible carrier fluid; and   (c) amplifying the one or more target polynucleotides in the amplification droplet;   wherein no reagents are selectively removed from the lysate droplet or from the amplification droplet prior to step (c).   
     
     
         2 . The method of  claim 1 , wherein (i) the amplification droplet has a volume of 0.001 to 1000 picoliters, or (ii) the amplification droplet has a diameter of between 0.1 microns to 1000 microns. 
     
     
         3 . The method of  claim 1 , wherein the lysate droplet contains lysate of a single cell. 
     
     
         4 . The method of  claim 1 , wherein step (b) comprises (i) merging the lysate droplet with a stream of fluid comprising the reagents for performing a nucleic acid amplification reaction, and (ii) forming the amplification droplet from the stream of fluid. 
     
     
         5 . The method of  claim 1 , wherein the reagents for performing a nucleic acid amplification reaction are added to the lysate droplet by droplet coalescence or picoinjection. 
     
     
         6 . The method of  claim 1 , wherein the one or more target polynucleotides are DNA. 
     
     
         7 . The method of  claim 1 , wherein the one or more target polynucleotides are RNA. 
     
     
         8 . The method of  claim 7 , wherein amplification comprises reverse transcription to produce a reverse transcription product. 
     
     
         9 . The method of  claim 1 , further comprising the step of detecting presence of an amplification product amplified in step (c). 
     
     
         10 . The method of  claim 9 , wherein detecting presence of an amplification product comprises sequencing the amplification product. 
     
     
         11 . The method of  claim 9 , wherein detecting presence of an amplification product comprises forming a double-emulsion comprising an amplification droplet within an outer droplet, and sorting the double-emulsion based on droplet size and fluorescence. 
     
     
         12 . The method of  claim 9 , further comprising the step of sorting the amplification droplet based on results of the detection step. 
     
     
         13 . The method of  claim 9 , further comprising determining the number or percentage of cells containing the one or more target polynucleotides based on results of the detection step. 
     
     
         14 . The method of  claim 9 , wherein amplification comprises extension of a primer comprising a capture sequence, and detecting presence of an amplification product comprises detecting the capture sequence in the amplification product. 
     
     
         15 . The method of  claim 1 , wherein one or more steps are performed under microfluidic control. 
     
     
         16 . The method of  claim 1 , wherein step (b) comprises applying an electrical field to the lysate droplet. 
     
     
         17 . The method of  claim 1 , wherein lysing the one or more polynucleotide-containing components comprises exposure to a protease, and further wherein the protease is inactivated prior to step (c). 
     
     
         18 . A method of amplifying one or more target polynucleotides, the method comprising:
 (a) lysing one or more polynucleotide-containing components from a sample with a protease in a droplet to form a lysate in a lysate droplet, wherein the lysate comprises the one or more target polynucleotides and the lysate droplet is in an immiscible carrier fluid;   (b) adding to the lysate reagents for performing a nucleic acid amplification reaction to form an amplification mixture in an amplification droplet, wherein the amplification droplet is in the immiscible carrier fluid; and   (c) amplifying the one or more target polynucleotides in the amplification droplet;   wherein the protease of step (a) is inactivated during or prior to step (c).   
     
     
         19 . The method of  claim 18 , wherein (i) the amplification droplet has a volume of 0.001 to 1000 picoliters, or (ii) the amplification droplet has a diameter of between 0.1 microns to 1000 microns. 
     
     
         20 . The method of  claim 18 , wherein the lysate droplet contains lysate of a single cell.