Selection and enrichment of proteins using in vitro compartmentalization
View Patent ↗Compositions and methods are provided for selection and enrichment of a target gene from a library of polynucleotide sequences such as might be formed from a genome or by random mutagenesis of a genetic sequence. The selection and enrichment occurs in aqueous droplets formed in an emulsion that compartmentalize individual polynucleotides from the library or a plurality of polynucleotides that may include polynucleotides not derived from the library, transcription and translation reagents and optionally additional chemical and enzyme reagents. The selection and enrichment method utilizes a polynucleotide adaptor which when ligated to the polynucleotide fragment enables amplification to occur in the presence of an adaptor specific primer.
1. An emulsion of a hydrophilic solution in a hydrophobic liquid, wherein the hydrophilic solution forms a plurality of droplets, each of the droplets containing:
(i) a mixture of enzymes with transcription and translation activity;
(ii) a polynucleotide adaptor with a sticky end; and
(iii) a polynucleotide fragment from a library of polynucleotide fragments, wherein the polynucleotide fragment (a) has a sticky end or a recognition site for an enzyme capable of cleaving the polynucleotide to create a sticky end; and (b) encodes a protein selected from the group consisting of RNA ligase, DNA ligase, restriction endonuclease, nicking endonuclease, homing endonuclease, tRNA synthetase, RNA polymerase and reverse transcriptase, which if active, permits the adaptor to become ligated to the polynucleotide fragment.
2. The emulsion according to claim 1 , further comprising (iv) one or more polynucleotide fragments not from the library, wherein the one or more polynucleotide fragments encodes a second protein.
3. The emulsion according to claim 2 , wherein the second protein is a restriction endonuclease or a nicking endonuclease.