Comparison of Commercially Available Thermostable DNA Polymerases with Reverse Transcriptase Activity in Coupled Reverse Transcription Polymerase Chain Reaction Assays
Reverse transcription polymerase chain reaction (RT-PCR) is an important tool for the detection of target RNA molecules and the assay of RNA pathogens. Coupled RT-PCR is performed with an enzyme mixture containing a reverse transcriptase and a thermostable DNA polymerase. To date, several biotechnol...
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2025-01-01
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| author | Evgeniya V. Smirnova Konstantin A. Blagodatskikh Ekaterina V. Barsova Dmitriy A. Varlamov Vladimir M. Kramarov Konstantin B. Ignatov |
| author_facet | Evgeniya V. Smirnova Konstantin A. Blagodatskikh Ekaterina V. Barsova Dmitriy A. Varlamov Vladimir M. Kramarov Konstantin B. Ignatov |
| author_sort | Evgeniya V. Smirnova |
| collection | DOAJ |
| description | Reverse transcription polymerase chain reaction (RT-PCR) is an important tool for the detection of target RNA molecules and the assay of RNA pathogens. Coupled RT-PCR is performed with an enzyme mixture containing a reverse transcriptase and a thermostable DNA polymerase. To date, several biotechnological companies offer artificial thermostable DNA polymerases with a built-in reverse transcriptase activity for use in the coupled RT-PCR instead of the enzyme mixtures. Here, we compared the artificial DNA polymerases and conventional enzyme mixtures for the RT-PCR by performing end-point and real-time RT-PCR assays using severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV2) RNA and endogenous mRNA molecules as templates. We found that the artificial enzymes were suitable for different RT-PCR applications, including SARS-CoV2 RNA detection but not for long-fragment RT-PCR amplification. |
| format | Article |
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| institution | DOAJ |
| issn | 2409-9279 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | MDPI AG |
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| series | Methods and Protocols |
| spelling | doaj-art-38dc842e24b2483c96a1fd314f3a041c2025-08-20T02:44:50ZengMDPI AGMethods and Protocols2409-92792025-01-01811110.3390/mps8010011Comparison of Commercially Available Thermostable DNA Polymerases with Reverse Transcriptase Activity in Coupled Reverse Transcription Polymerase Chain Reaction AssaysEvgeniya V. Smirnova0Konstantin A. Blagodatskikh1Ekaterina V. Barsova2Dmitriy A. Varlamov3Vladimir M. Kramarov4Konstantin B. Ignatov5Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, RussiaLaboratory of Molecular Oncology, Pirogov Russian National Research Medical University, 117513 Moscow, RussiaShemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, RussiaSyntol JSC, 127550 Moscow, RussiaVavilov Institute of General Genetics, Russian Academy of Sciences, 119991 Moscow, RussiaVavilov Institute of General Genetics, Russian Academy of Sciences, 119991 Moscow, RussiaReverse transcription polymerase chain reaction (RT-PCR) is an important tool for the detection of target RNA molecules and the assay of RNA pathogens. Coupled RT-PCR is performed with an enzyme mixture containing a reverse transcriptase and a thermostable DNA polymerase. To date, several biotechnological companies offer artificial thermostable DNA polymerases with a built-in reverse transcriptase activity for use in the coupled RT-PCR instead of the enzyme mixtures. Here, we compared the artificial DNA polymerases and conventional enzyme mixtures for the RT-PCR by performing end-point and real-time RT-PCR assays using severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV2) RNA and endogenous mRNA molecules as templates. We found that the artificial enzymes were suitable for different RT-PCR applications, including SARS-CoV2 RNA detection but not for long-fragment RT-PCR amplification.https://www.mdpi.com/2409-9279/8/1/11reverse transcriptasethermostable DNA polymerasereverse transcription polymerase chain reactionSARS-CoV2beta-2-microglobulin mRNA<i>GAPDH</i> mRNA |
| spellingShingle | Evgeniya V. Smirnova Konstantin A. Blagodatskikh Ekaterina V. Barsova Dmitriy A. Varlamov Vladimir M. Kramarov Konstantin B. Ignatov Comparison of Commercially Available Thermostable DNA Polymerases with Reverse Transcriptase Activity in Coupled Reverse Transcription Polymerase Chain Reaction Assays Methods and Protocols reverse transcriptase thermostable DNA polymerase reverse transcription polymerase chain reaction SARS-CoV2 beta-2-microglobulin mRNA <i>GAPDH</i> mRNA |
| title | Comparison of Commercially Available Thermostable DNA Polymerases with Reverse Transcriptase Activity in Coupled Reverse Transcription Polymerase Chain Reaction Assays |
| title_full | Comparison of Commercially Available Thermostable DNA Polymerases with Reverse Transcriptase Activity in Coupled Reverse Transcription Polymerase Chain Reaction Assays |
| title_fullStr | Comparison of Commercially Available Thermostable DNA Polymerases with Reverse Transcriptase Activity in Coupled Reverse Transcription Polymerase Chain Reaction Assays |
| title_full_unstemmed | Comparison of Commercially Available Thermostable DNA Polymerases with Reverse Transcriptase Activity in Coupled Reverse Transcription Polymerase Chain Reaction Assays |
| title_short | Comparison of Commercially Available Thermostable DNA Polymerases with Reverse Transcriptase Activity in Coupled Reverse Transcription Polymerase Chain Reaction Assays |
| title_sort | comparison of commercially available thermostable dna polymerases with reverse transcriptase activity in coupled reverse transcription polymerase chain reaction assays |
| topic | reverse transcriptase thermostable DNA polymerase reverse transcription polymerase chain reaction SARS-CoV2 beta-2-microglobulin mRNA <i>GAPDH</i> mRNA |
| url | https://www.mdpi.com/2409-9279/8/1/11 |
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