Combined multiple-displacement amplification and pcr in an emulsion microdroplet

Abstract

The methods and systems described herein provide an improved emulsion droplet based nucleic acid amplification method, which allows nucleic acids contained in biological systems to be detected, quantitated and/or sorted based on their sequence as detected with nucleic acid amplification techniques, e.g., polymerase chain reaction (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.

1 - 11 . (canceled) 
     
     
         12 . A nucleic acid amplification method including:
 flowing a miscible phase fluid solution of nucleic acids and amplification reagents in a channel of a microfluidic device;   contacting the miscible phase fluid solution of nucleic acids and amplification reagents with an immiscible phase fluid, wherein the contacting of the miscible phase fluid solution of nucleic acids and amplification reagents with the immiscible phase fluid results in the formation of miscible phase microdroplets surrounded by the immiscible phase fluid;   flowing the miscible phase microdroplets surrounded by the immiscible phase fluid in a channel of a microfluidic device;   contacting the miscible phase microdroplets surrounded by the immiscible phase fluid with a miscible phase carrier fluid, wherein the contacting of the miscible phase microdroplets surrounded by the immiscible phase fluid with the miscible phase carrier fluid results in the formation of multiple-emulsion microdroplets, each multiple-emulsion microdroplet including a miscible phase microdroplet surrounded by the immiscible phase fluid, wherein the immiscible phase fluid is surrounded by the miscible phase carrier fluid;   subjecting the multiple-emulsion microdroplets to amplification conditions sufficient to result in amplification of a target nucleic acid when present in the miscible phase fluid solution of nucleic acids; and   detecting an amplification product resulting from the amplification of the target nucleic acid when present in the miscible phase fluid solution of nucleic acids.   
     
     
         13 . The method of  claim 12 , wherein the miscible phase carrier fluid is a buffered aqueous phase carrier fluid. 
     
     
         14 . The method of  claim 12 , wherein the miscible phase fluid of the miscible phase fluid solution and the miscible phase carrier fluid are the same. 
     
     
         15 . The method of  claim 12 , wherein subjecting the multiple-emulsion microdroplets to amplification conditions includes subjecting the multiple-emulsion microdroplets to polymerase chain reaction (PCR) conditions. 
     
     
         16 . The method of  claim 12 , wherein subjecting the multiple-emulsion microdroplets to amplification conditions includes subjecting the multiple-emulsion microdroplets to isothermal amplification conditions. 
     
     
         17 . The method of  claim 16 , wherein the isothermal amplification conditions are selected from loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase-dependent amplification (HDA), and nicking enzyme amplification reaction (NEAR). 
     
     
         18 . The method of  claim 12 , including detectably labeling the amplification product subsequent to amplification. 
     
     
         19 . The method of  claim 12 , wherein the amplification reagents include detectably labeled primers and/or probes. 
     
     
         20 . The method of  claim 12 , including detectably labeling the amplification product with a fluorescent label and sorting the multiple-emulsion microdroplets via fluorescence activating cell sorting (FACS). 
     
     
         21 . The method of  claim 12 , including adjusting the composition of the miscible phase fluid solution by adjusting the composition of the miscible phase carrier fluid. 
     
     
         22 . The method of  claim 12 , including detectably labeling the amplification product by adding a detectable label to the miscible phase carrier fluid, wherein the detectable label diffuses from the miscible phase carrier fluid, through the immiscible phase fluid, and into the miscible phase fluid solution. 
     
     
         23 . The method of  claim 12 , wherein the multiple-emulsion microdroplets are first multiple-emulsion microdroplets, and the method includes adding a reagent to the first multiple-emulsion microdroplets, wherein the adding includes encapsulating the first multiple-emulsion microdroplets in second multiple-emulsion microdroplets including the reagent and rupturing the first multiple-emulsion microdroplets within the second multiple-emulsion microdroplets to bring the reagent into contact with the contents of the first multiple-emulsion microdroplets. 
     
     
         24 . The method of  claim 12 , wherein the multiple-emulsion microdroplets are second multiple-emulsion microdroplets, and the method includes adding a reagent to the second multiple-emulsion microdroplets, wherein the adding includes encapsulating first multiple-emulsion microdroplets including the reagent in the second multiple-emulsion microdroplets and rupturing the first multiple-emulsion microdroplets within the second multiple-emulsion microdroplets to bring the reagent into contact with the contents of the second multiple-emulsion microdroplets.