PCR-Activated Sorting (PAS)

Abstract

The methods described herein, referred to as PCR-Activated Sorting (PAS), allow nucleic acids contained in biological systems to be sorted based on their sequence as detected with nucleic acid amplification techniques, e.g., PCR. The nucleic acids can be free floating or contained within living or nonliving structures, including particles, viruses, and cells. The nucleic acids can include, e.g., DNA or RNA. Systems and devices for use in practicing methods of the invention are also provided.

What is claimed is: 
     
         1 . A method for sorting samples comprising nucleic acids, the method comprising:
 encapsulating a sample comprising nucleic acids in a plurality of microdroplets, each microdroplet comprising an aqueous phase fluid in an immiscible phase carrier fluid;   introducing polymerase chain reaction (PCR) reagents and a plurality of PCR primers into the microdroplets;   incubating the microdroplets under conditions sufficient for PCR amplification to produce PCR amplification products, wherein the plurality of PCR primers include one or more primers that each hybridize to one or more oligonucleotides;   introducing a detection component into the microdroplets either before or after the incubating;   detecting the presence or absence of the PCR amplification products by detection of the detection component, wherein detection of the detection component indicates the presence of PCR amplification products;   after the incubating, and before or after the detecting, positioning the microdroplets in an aqueous phase carrier fluid to provide aqueous phase-in-immiscible phase-in aqueous phase microdroplets; and   sorting the aqueous phase-in-immiscible phase-in aqueous phase microdroplets based on detection of the detection component, wherein the sorting separates aqueous phase-in-immiscible phase-in aqueous phase microdroplets comprising the PCR amplification products, when present, from aqueous phase-in-immiscible phase-in aqueous phase microdroplets which do not comprise the PCR amplification products, wherein one or more steps are performed under microfluidic control.   
     
     
         2 . The method of  claim 1 , wherein the detection component is introduced before the incubating. 
     
     
         3 . The method of  claim 1 , wherein the detection component is introduced after the incubating. 
     
     
         4 . The method of any one of  claims 1 - 3 , wherein the sorting comprises sorting the aqueous phase-in-immiscible phase-in aqueous phase microdroplets via Fluorescence-Activated Cell Sorting (FACS). 
     
     
         5 . The method of any one of  claims 1 - 4 , wherein the sorting comprises sorting the aqueous phase-in-immiscible phase-in aqueous phase microdroplets microfluidically. 
     
     
         6 . The method of  claim 5 , wherein sorting the aqueous phase-in-immiscible phase-in aqueous phase microdroplets microfluidically comprises sorting the aqueous phase-in-immiscible phase-in aqueous phase microdroplets via dielectrophoresis. 
     
     
         7 . The method of  claim 5 , wherein sorting the microdroplets microfluidically comprises sorting the microdroplets via activation of one or more microfluidic valves. 
     
     
         8 . The method of any one of  claims 1 - 7 , wherein the nucleic acids are encapsulated such that each microdroplet independently comprises not more than one nucleic acid. 
     
     
         9 . The method of any one of  claims 1 - 8 , wherein the nucleic acids are comprised by a plurality of cells in the sample. 
     
     
         10 . The method of  claim 9 , wherein the method comprises introduction of a cell lysis reagent or incubating the microdroplets under conditions effective for cell lysis prior to introducing the PCR reagents and the plurality of PCR primers into the microdroplets. 
     
     
         11 . The method of  claim 9  or  10 , wherein the cells are encapsulated such that each microdroplet independently comprises not more than one cell. 
     
     
         12 . The method of any one of  claims 1 - 7 , wherein the nucleic acids are comprised by a plurality of viruses. 
     
     
         13 . The method of  claim 12 , wherein the method comprises introduction of a viral lysis reagent or incubating the microdroplets under conditions effective for viral lysis prior to introducing the PCR reagents and the plurality of PCR primers into the microdroplets. 
     
     
         14 . The method of  claim 12  or  13 , wherein the viruses are encapsulated such that each microdroplet independently comprises not more than one virus. 
     
     
         15 . The method of claim any one of  claims 1 - 14 , comprising recovering the contents of one or more of the sorted aqueous phase-in-immiscible phase-in aqueous phase microdroplets by rupturing the one or more sorted aqueous phase-in-immiscible phase-in aqueous phase microdroplets. 
     
     
         16 . A method for enriching for a target nucleic acid, the method comprising:
 encapsulating a sample comprising nucleic acids in a plurality of microdroplets, each microdroplet comprising a first aqueous phase fluid in an immiscible phase carrier fluid;   introducing polymerase chain reaction (PCR) reagents and a plurality of PCR primers into the microdroplets;   incubating the microdroplets under conditions sufficient for PCR amplification to produce one or more PCR amplification products, wherein the plurality of PCR primers include one or more primers that each hybridize to one or more oligonucleotides comprised by the target nucleic acid, and wherein the PCR amplification products do not comprise the entire target nucleic acid;   introducing a detection component into the microdroplets either before or after the incubating;   detecting the presence or absence of the PCR amplification products by detection of the detection component, wherein detection of the detection component indicates the presence of PCR amplification products and the target nucleic acid; and   sorting the microdroplets based on detection of the detection component, wherein the sorting separates microdroplets comprising the PCR amplification products and the target nucleic acid, when present, from microdroplets which do not comprise the PCR amplification products and the target nucleic acid; and   pooling the target nucleic acids from the sorted microdroplets to provide an enriched pool of the target nucleic acids, when present, wherein one or more steps are performed under microfluidic control.   
     
     
         17 . The method of  claim 16 , wherein the detection component is introduced before the incubating. 
     
     
         18 . The method of  claim 16 , wherein the detection component is introduced after the incubating. 
     
     
         19 . The method of any one of  claims 16 - 18 , wherein after the incubating, and before or after the detecting, the method comprises positioning the microdroplets in an aqueous phase carrier fluid to provide aqueous phase-in-immiscible phase-in aqueous phase microdroplets, wherein the sorting comprises sorting the aqueous phase-in-immiscible phase-in aqueous phase microdroplets based on detection of the detection component,
 wherein the sorting separates aqueous phase-in-immiscible phase-in aqueous phase microdroplets comprising the PCR amplification products and the target nucleic acid, when present, from aqueous phase-in-immiscible phase-in aqueous phase microdroplets which do not comprise the PCR amplification products and the target nucleic acid, and   wherein the pooling comprises pooling the target nucleic acids from the sorted aqueous phase-in-immiscible phase-in aqueous phase microdroplets to provide an enriched pool of the target nucleic acids, when present, wherein one or more steps are performed under microfluidic control.   
     
     
         20 . The method of  claim 19 , wherein the sorting comprises sorting the aqueous phase-in-immiscible phase-in aqueous phase microdroplets via Fluorescence-Activated Cell Sorting (FACS). 
     
     
         21 . The method of any one of  claims 16 - 20 , wherein the sorting comprises sorting the aqueous phase-in-immiscible phase-in aqueous phase microdroplets microfluidically. 
     
     
         22 . The method of  claim 21 , wherein sorting the aqueous phase-in-immiscible phase-in aqueous phase microdroplets microfluidically comprises sorting the aqueous phase-in-immiscible phase-in aqueous phase microdroplets via dielectrophoresis. 
     
     
         23 . The method of  claim 21 , wherein sorting the aqueous phase-in-immiscible phase-in aqueous phase microdroplets microfluidically comprises sorting the aqueous phase-in-immiscible phase-in aqueous phase microdroplets via activation of one or more microfluidic valves. 
     
     
         24 . The method of any one of  claims 16 - 23 , wherein the nucleic acids are encapsulated such that each microdroplet independently comprises not more than one nucleic acid. 
     
     
         25 . The method of any one of  claims 16 - 24 , wherein the nucleic acids are comprised by a plurality of cells in the sample. 
     
     
         26 . The method of  claim 25 , wherein the method comprises introduction of a cell lysis reagent or incubating the microdroplets under conditions effective for cell lysis prior to introducing the PCR reagents and the plurality of PCR primers into the microdroplets. 
     
     
         27 . The method of  claim 25  or  26 , wherein the cells are encapsulated such that each microdroplet independently comprises not more than one cell. 
     
     
         28 . The method of any one of  claims 16 - 24 , wherein the nucleic acids are comprised by a plurality of viruses. 
     
     
         29 . The method of  claim 28 , wherein the method comprises introduction of a viral lysis reagent or incubating the microdroplets under conditions effective for viral lysis prior to introducing the PCR reagents and the plurality of PCR primers into the microdroplets. 
     
     
         30 . The method of  claim 28  or  29 , wherein the viruses are encapsulated such that each microdroplet independently comprises not more than one virus. 
     
     
         31 . The method of claim any one of  claims 16 - 30 , comprising recovering the contents of one or more of the sorted microdroplets or aqueous phase-in-immiscible phase-in aqueous phase microdroplets by rupturing the one or more sorted microdroplets or aqueous phase-in-immiscible phase-in aqueous phase microdroplets. 
     
     
         32 . The method of any one of  claims 16 - 31 , wherein the target nucleic acid sequence is greater than 10 kilobases in length. 
     
     
         33 . The method of  claim 32 , wherein the target nucleic acid sequence is greater than 100 kilobases in length. 
     
     
         34 . The method of  claim 33 , wherein the target nucleic acid sequence is greater than 1 megabase in length. 
     
     
         35 . The method of any one of  claims 16 - 34 , comprising sequencing the target nucleic acid sequence from the enriched pool of target nucleic acid sequences. 
     
     
         36 . The method of any one of  claims 16 - 35 , wherein the plurality of PCR primers comprises a first set of primers designed to amplify a first region of the target nucleic acid and a second set of primers designed to amplify a second region of the target nucleic acid, wherein the first and second region are separated by at least 10 kilobases in the target nucleic acid.