Engineering of novel DNA polymerase variants for single enzyme quantitative multiplex Reverse Transcription-PCR

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. Well-established 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 the first DNA pols that 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. Overall design: SARS-CoV-2 (in vitro RNA, purchased from European Commission Joint Research Centre (EURM-019)) was used for RT-PCR catalsyed by RT-Taq, Mut_RT, Taq pol V2, Taq pol V3 and Taq pol V2 IL. Respective cDNA was used to prepare DNA amplicon libraries which were analyzed by NGS