Comparison of Commercially Available Thermostable DNA Polymerases with Reverse Transcriptase Activity in Coupled Reverse Transcription Polymerase Chain Reaction Assays

Abstract

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.

Keywords: GAPDH mRNA; SARS-CoV2; beta-2-microglobulin mRNA; reverse transcriptase; reverse transcription polymerase chain reaction; thermostable DNA polymerase.

Figure 1

End-point RT-PCR assays with RevTaq, OmniTaq 2, and ReverHotTaq DNA polymerases and OneTaq One-Step Enzyme Mix. The indicated DNA polymerases and OneTaq enzyme mixture were used for the RT-PCR amplification of 105 bp, 203 bp, and 317 bp fragments (indicated by arrows) of the human beta-2-microglobulin mRNA sequence. The reaction mixtures contained a human total RNA as a template: 10 ng—lane 1; 1 ng—lane 2; 100 pg—lane 3; 10 pg—lane 4; no template control (NTC)—lane 5.

Figure 1

End-point RT-PCR assays with RevTaq, OmniTaq 2, and ReverHotTaq DNA polymerases and OneTaq One-Step Enzyme Mix. The indicated DNA polymerases and OneTaq enzyme mixture were used for the RT-PCR amplification of 105 bp, 203 bp, and 317 bp fragments (indicated by arrows) of the human beta-2-microglobulin mRNA sequence. The reaction mixtures contained a human total RNA as a template: 10 ng—lane 1; 1 ng—lane 2; 100 pg—lane 3; 10 pg—lane 4; no template control (NTC)—lane 5.

Figure 2

Real-time RT-qPCR with EvaGreen intercalating dye and melting curves of amplicons. The RT-qPCR assays of human GAPDH mRNA in the total RNA were carried out with the indicated enzymes: ReverHotTaq (A), RevTaq (B), and OmniTaq2 (C) DNA polymerases and OneTube RT-PCRMix (D). The reaction mixtures contained 1 ng (brown curves), 0.1 ng (beige curves), or 10 pg (green curves) of human total RNA per reaction, or no template NTC (gray curves). Melting curves demonstrate the yields of specific (melting peaks over 80 °C) and non-specific (melting peaks below 80 °C) amplicons.

Figure 3

Real-time RT-qPCR with TaqMan probe. The RT-qPCR assays of SARS-CoV-2 viral RNA were carried out with the indicated enzymes: ReverHotTaq (A), RevTaq (C), and OmniTaq2 (D) DNA polymerases and OneTube RT-PCRMix (B). The reaction mixtures contained 10 times diluted (brown curves), 50 times diluted (beige curves), or 250 times diluted (green curves) sample of RNA isolated from SARS-CoV-2-positive nasopharyngeal swabs as a template or no template NTC (gray curves).