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Real Time RT-PCR02:57

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Comparison of Commercially Available Thermostable DNA Polymerases with Reverse Transcriptase Activity in Coupled

Evgeniya V Smirnova1, Konstantin A Blagodatskikh2,3, Ekaterina V Barsova1,4

  • 1Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia.

Methods and Protocols
|February 25, 2025
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Summary

Artificial DNA polymerases with built-in reverse transcriptase activity are effective for SARS-CoV2 RNA detection via reverse transcription polymerase chain reaction (RT-PCR). However, they are not suitable for amplifying long RNA fragments.

Keywords:
GAPDH mRNASARS-CoV2beta-2-microglobulin mRNAreverse transcriptasereverse transcription polymerase chain reactionthermostable DNA polymerase

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Area of Science:

  • Molecular Biology
  • Virology
  • Biotechnology

Background:

  • Reverse transcription polymerase chain reaction (RT-PCR) is crucial for detecting RNA targets and pathogens.
  • Conventional RT-PCR uses enzyme mixtures of reverse transcriptase and thermostable DNA polymerase.
  • Biotechnology companies now offer artificial DNA polymerases with integrated reverse transcriptase activity for coupled RT-PCR.

Purpose of the Study:

  • To compare the performance of artificial DNA polymerases against conventional enzyme mixtures in RT-PCR assays.
  • To evaluate the suitability of artificial enzymes for detecting SARS-CoV2 RNA and endogenous mRNA.
  • To assess the utility of artificial enzymes in both end-point and real-time RT-PCR applications.

Main Methods:

  • Performed end-point and real-time RT-PCR assays.
  • Utilized severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV2) RNA as a template.
  • Employed endogenous mRNA molecules as additional templates for comparison.

Main Results:

  • Artificial DNA polymerases demonstrated suitability for various RT-PCR applications.
  • Detection of SARS-CoV2 RNA was successful using the artificial enzymes.
  • Artificial enzymes were found to be unsuitable for long-fragment RT-PCR amplification.

Conclusions:

  • Artificial thermostable DNA polymerases with built-in reverse transcriptase activity are viable alternatives for specific RT-PCR applications, including viral RNA detection.
  • These artificial enzymes offer convenience but have limitations, particularly in amplifying extended RNA fragments.
  • Further research may be needed to optimize artificial enzymes for broader RT-PCR applications.