Directed evolution method

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 . A nucleic acid processing (NAP) enzyme identified by a method comprising the steps of:
 (a) providing a pool of nucleic acids comprising members encoding a NAP enzyme or a variant of a NAP enzyme;   (b) subdividing the pool of nucleic acids into compartments, such that each compartment comprises a nucleic acid member of the pool together with the NAP enzyme or variant encoded by the nucleic acid member;   (c) allowing nucleic acid processing to occur; and   (d) detecting processing of the nucleic acid member by the NAP enzyme, whereby a NAP enzyme is selected.   
     
     
         2 . The NAP enzyme of  claim 1 , wherein said NAP enzyme is a variant of a known NAP enzyme, and wherein said variant has greater thermostability than said known NAP enzyme. 
     
     
         3 . The NAP enzyme of  claim 1 , wherein said NAP enzyme is a variant of a known NAP enzyme, and wherein said variant is inhibited to a lesser extent by heparin than is said known NAP enzyme. 
     
     
         4 . The NAP enzyme of  claim 3  which is a Taq polymerase active at a concentration of 0.083 units/μl or more of heparin. 
     
     
         5 . The NAP enzyme of  claim 1  which is a Taq polymerase active at a concentration of 0.083 units/μl or more of heparin. 
     
     
         6 . The NAP enzyme of  claim 1  which is a replicase enzyme that extends a primer having a 3′ mismatch. 
     
     
         7 . The NAP enzyme of  claim 1  which is a replicase enzyme that extends a primer having a 3′ unnatural base. 
     
     
         8 . The NAP enzyme of  claim 7  which is capable of extending a primer having a 3′ terminal base comprising 5-nitroindole or 3-carboxyamide-5-nitroindole. 
     
     
         9 . The NAP enzyme of  claim 1  which is a replicase enzyme variant of a known replicase enzyme, wherein said variant incorporates α-thio dNTPs as nucleotide substrates more efficiently than said known replicase enzyme. 
     
     
         10 . The NAP enzyme of  claim 1  which is a replicase enzyme variant of a known replicase enzyme, wherein said variant replicates a substrate 23 kb in size in the absence of processivity factors or a 3′-5′ exonuclease proof-reading domain. 
     
     
         11 . The NAP enzyme of  claim 6  in which the 3′ mismatch is a 3′ purine-purine mismatch or a 3′ pyrimidine-pyrimidine mismatch. 
     
     
         12 . The NAP enzyme of  claim 6  in which the 3′ mismatch is an A-G mismatch or in which the 3′ mismatch is a C-C mismatch. 
     
     
         13 . A Taq polymerase mutant comprising a mutation selected from the group consisting of G84A, D144G, K314R, E520G, A608V and E742G. 
     
     
         14 . A Taq polymerase mutant comprising G84A, D144G, K314R, E520G, A608V and E742G mutations.