Methods of increasing the concentration of a nucleic acid

Abstract

We describe a method of selecting an enzyme having replicase activity, the method comprising the steps of: (a) providing a pool of nucleic acids comprising members each encoding a replicase or a variant of the replicase; (b) subdividing the pool of nucleic acids into compartments, such that each compartment comprises a nucleic acid member of the pool together with the replicase or variant encoded by the nucleic acid member; (c) allowing nucleic acid replication to occur; and (d) detecting amplification of the nucleic acid member by the replicase. Methods for selecting agents capable of modulating replicase activity, and for selecting interacting polypeptides are also disclosed.

1 . (canceled) 
     
     
         2 . A method for increasing concentration of a nucleic acid molecule comprising the steps of:
 (a) forming microcapsules from a thermostable water-in-oil emulsion, wherein a plurality of microcapsules include a nucleic acid molecule, a bead capable of being linked to the nucleic acid molecule, and an aqueous solution comprising components necessary to perform PCR nucleic acid amplification, including a thermostable polymerase, wherein the emulsion has a density of greater than 10 9  microcapsules per ml;   (b) amplifying the nucleic acid molecule in the microcapsules to form amplified copies of the nucleic acid molecule; thereby increasing the concentration of the nucleic acid molecule; and   (c) sorting the bead with the captured amplification copies for sequencing.   
     
     
         3 . The method of  claim 2  wherein the amplified copies are captured to the beads in the microcapsules. 
     
     
         4 . The method of  claim 2  wherein the nucleic acid amplification is performed using a method selected from the group consisting of ligase chain reaction, self sustained sequence replication, reverse transcriptase-polymerase chain reaction, nucleic acid sequence-based amplification, and strand displacement amplification. 
     
     
         5 . The method of  claim 2  wherein the nucleic acid amplification is performed using polymerase chain reaction. 
     
     
         6 . The method of  claim 2  wherein the water-in-oil emulsion includes an emulsion stabilizer. 
     
     
         7 . The method of  claim 6  wherein the emulsion stabilizer is a non-ionic surfactant. 
     
     
         8 . The method of  claim 7  wherein the emulsion stabilizer is selected from the group consisting of sorbitan monooleate, polyoxyethylenesorbitan monooleate, and octylphenoxypolyethoxyethanol. 
     
     
         9 . The method of  claim 8  wherein the emulsion stabilizer is selected from the group consisting of Span80, Tween80, and TritonX-100. 
     
     
         10 . The method of  claim 6  wherein the emulsion stabilizer is an anionic surfactant. 
     
     
         11 . The method of  claim 10  wherein the emulsion stabilizer is selected from the group consisting of sodium cholate, sodium taurocholate, and sodium deoxycholate. 
     
     
         12 . The method of  claim 2  wherein the nucleic acid molecule is genomic DNA or cDNA. 
     
     
         13 . The method of  claim 2  wherein the nucleic acid molecule comprises a biotin tag. 
     
     
         14 . The method of  claim 2  wherein the bead comprises a coating of streptavidin or oligonucleotides. 
     
     
         15 . The method of  claim 2  wherein the bead is selected from the group consisting of polystyrene and magnetic beads. 
     
     
         16 . The method of  claim 2  wherein the water-in-oil phase ratio is between 1:2 and 1:4. 
     
     
         17 . The method of  claim 2  wherein a plurality of microcapsules when formed each contains on average one or less than one nucleic acid molecule. 
     
     
         18 . The method of  claim 2  wherein a plurality of microcapsules when formed each contains on average between 5 and 1000 nucleic acid molecules. 
     
     
         19 . The method of  claim 2  wherein the emulsion is stable for 20 cycles of PCR. 
     
     
         20 . The method of  claim 2  wherein the nucleic acid is delivered to the microcapsule by delivering a cell containing the nucleic acid to the microcapsule.