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In theory, organocatalysts capable of in situ error correction of their own molecular architecture could then be envisaged.
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Figure 4 depicts the procedure of in situ error-prone PPCP.
This dsDNA PCR product can be used as the primer pair in subsequent in situ error-prone mutagenic PPCP.
> -wrap-foot> We used in situ error-prone PPCP to improve the activities of two cellulases from T. maritima.
Mutant libraries of celA, celB and xynA1, respectively, were constructed by in situ error-prone mutagenic PPCP as described above.
These results confirm the in situ error-prone PPCP procedure in being able to rapidly mutate a target gene.
The in situ error-prone mutagenic PPCP can be coupled to a screening or selection procedure, facilitating directed evolution of the target gene or protein.
To experimentally demonstrate the in situ error-prone PPCP concept, we chose a pair of identical expression vectors comprising different selection markers.
Screening of these libraries resulted in mutant proteins with desired properties, demonstrating the usefulness of in situ error-prone PPCP for creating random mutagenesis libraries for directed evolution.
Finally, when an error-prone DNA polymerase is used, PPCP can be used for in situ mutagenesis to create random mutagenesis libraries (in situ error-prone mutagenic PPCP).
The results demonstrated that in situ error-prone PPCP is an efficient random mutagenesis method for directed evolution of genes and proteins.
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