期刊
ACS SYNTHETIC BIOLOGY
卷 9, 期 7, 页码 1873-1881出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.0c00236
关键词
parallel mutagenesis; microfluidics; droplet sorting; polymerase evolution
资金
- National Science Foundation [MCB: 1946312]
- Genomics High Throughput Facility Shared Resource of the Cancer Center Support Grant at the University of California, Irvine [P30CA-062203]
- NIH shared instrumentation grants [1S10RR025496-01, 1S10OD010794-01, 1S10OD021718-01]
Most DNA polymerase libraries sample unknown portions of mutational space and are constrained by the limitations of random mutagenesis. Here we describe a programmed allelic mutagenesis (PAM) strategy to comprehensively evaluate all possible single-point mutations in the entire catalytic domain of a replicative DNA polymerase. By applying the PAM strategy with ultrafast high-throughput screening, we show how DNA polymerases can be mapped for allelic mutations that exhibit enhanced activity for unnatural nucleic acid substrates. We suggest that comprehensive missense mutational scans may aid the discovery of specificity determining residues that are necessary for reprogramming the biological functions of natural DNA polymerases.
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