ORIGINAL RESEARCH article
Front. Bioeng. Biotechnol.
Sec. Synthetic Biology
Volume 13 - 2025 | doi: 10.3389/fbioe.2025.1624735
Live Culture-Based qPCR Screening of Taq DNA Polymerase Variants for Resistance to PCR Inhibitors
Provisionally accepted
- 1DNA Polymerase Technology, Inc., St. Louis, United States
- 2Biochemistry and Molecular Biophysics, Washington University School of Medicine,, Saint Louis, United States
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We present a fast and convenient method for direct real-time PCR-based screening of mutagenized libraries of Taq DNA polymerase for desired enzyme phenotypes, such as resistance to inhibitors. The novel procedure, "live culture PCR" (LC-PCR), is based on the finding that total unprocessed bacterial culture expressing Taq pol, Klentaq1, or their variants can catalyze efficient amplification of both endogenous and exogenous DNA. Thus, the procedure eliminates any enzyme extraction, cell manipulation, or lysis steps prior to PCR. A library to be screened need not even be plated initially; rather, a liquid culture of it can be diluted to about 5-10 cells per well before growth. We demonstrate the LC-PCR method by successful screening of a mutagenized library to identify Taq and Klentaq1 mutants which are highly resistant to PCR inhibitors found in food, blood, plant, and soil samples.
Keywords: Life Culture PCR (LC-PCR), Enzyme screening, Mutagenesis, PCR inhibitor, Taq Polymerase
Received: 07 May 2025; Accepted: 08 Aug 2025.
Copyright: © 2025 Kermekchiev, Zhang and Barnes. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Zhian Zhang, DNA Polymerase Technology, Inc., St. Louis, United States
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