PCR-Activated Sorting (PAS)

摘要

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.

主权项

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.