Engineering of novel DNA polymerase variants for single enzyme quantitative multiplex reverse transcription-PCR
Abstract Reverse transcription polymerase chain reaction (RT-PCR) has evolved as a widely used approach in biotechnology and molecular diagnostics. It represents a powerful tool for amplifying and analysing RNA molecules and has therefore found widespread applications in profiling gene expression, v...
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| Format: | Article |
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Nature Portfolio
2025-07-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-025-10211-x |
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| author | Luisa B. Huber Virginie Marchand Melike Somtürk Silke Müller Andreas Marx |
| author_facet | Luisa B. Huber Virginie Marchand Melike Somtürk Silke Müller Andreas Marx |
| author_sort | Luisa B. Huber |
| collection | DOAJ |
| description | Abstract Reverse transcription polymerase chain reaction (RT-PCR) has evolved as a widely used approach in biotechnology and molecular diagnostics. It represents a powerful tool for amplifying and analysing RNA molecules and has therefore found widespread applications in profiling gene expression, viral detection and the diagnosis of various diseases. Wellestablished methodologies use viral reverse transcriptases (RTs) to transcribe RNA to cDNA and thermostable DNA polymerases (DNA pols) to amplify the resulting target sequence by PCR. This study reports on the development of novel Thermus aquaticus DNA polymerase I (Taq pol) variants that each are able to catalyse both steps simultaneously in a single tube without the need of viral RTs. In combination with their excellent thermostability (up to 95 °C), the novel Taq pol variants are suitable for employment in dye- or probe-based RNA detection methods. Moreover, the herein reported Taq pol variants are capable of performing multiplex detection of various RNA targets in a single tube with a single enzyme. Thus, discovery marks a significant advancement of current RT-PCR approaches and contributes simplifying and reducing costs in molecular diagnostics. |
| format | Article |
| id | doaj-art-848b47c54d3a463293476d882acebb6a |
| institution | Kabale University |
| issn | 2045-2322 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj-art-848b47c54d3a463293476d882acebb6a2025-08-20T04:01:51ZengNature PortfolioScientific Reports2045-23222025-07-0115111810.1038/s41598-025-10211-xEngineering of novel DNA polymerase variants for single enzyme quantitative multiplex reverse transcription-PCRLuisa B. Huber0Virginie Marchand1Melike Somtürk2Silke Müller3Andreas Marx4Department of Chemistry, Konstanz Research School Chemical Biology, University of KonstanzSMP IBSLor, Epitranscriptomics and RNA Sequencing Core Facility, Université de LorraineDepartment of Chemistry, University of KonstanzDepartment of Biology, Screening Centre, University of KonstanzDepartment of Chemistry, Konstanz Research School Chemical Biology, University of KonstanzAbstract Reverse transcription polymerase chain reaction (RT-PCR) has evolved as a widely used approach in biotechnology and molecular diagnostics. It represents a powerful tool for amplifying and analysing RNA molecules and has therefore found widespread applications in profiling gene expression, viral detection and the diagnosis of various diseases. Wellestablished methodologies use viral reverse transcriptases (RTs) to transcribe RNA to cDNA and thermostable DNA polymerases (DNA pols) to amplify the resulting target sequence by PCR. This study reports on the development of novel Thermus aquaticus DNA polymerase I (Taq pol) variants that each are able to catalyse both steps simultaneously in a single tube without the need of viral RTs. In combination with their excellent thermostability (up to 95 °C), the novel Taq pol variants are suitable for employment in dye- or probe-based RNA detection methods. Moreover, the herein reported Taq pol variants are capable of performing multiplex detection of various RNA targets in a single tube with a single enzyme. Thus, discovery marks a significant advancement of current RT-PCR approaches and contributes simplifying and reducing costs in molecular diagnostics.https://doi.org/10.1038/s41598-025-10211-x |
| spellingShingle | Luisa B. Huber Virginie Marchand Melike Somtürk Silke Müller Andreas Marx Engineering of novel DNA polymerase variants for single enzyme quantitative multiplex reverse transcription-PCR Scientific Reports |
| title | Engineering of novel DNA polymerase variants for single enzyme quantitative multiplex reverse transcription-PCR |
| title_full | Engineering of novel DNA polymerase variants for single enzyme quantitative multiplex reverse transcription-PCR |
| title_fullStr | Engineering of novel DNA polymerase variants for single enzyme quantitative multiplex reverse transcription-PCR |
| title_full_unstemmed | Engineering of novel DNA polymerase variants for single enzyme quantitative multiplex reverse transcription-PCR |
| title_short | Engineering of novel DNA polymerase variants for single enzyme quantitative multiplex reverse transcription-PCR |
| title_sort | engineering of novel dna polymerase variants for single enzyme quantitative multiplex reverse transcription pcr |
| url | https://doi.org/10.1038/s41598-025-10211-x |
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