Strand Displacement Activity of Mimiviral Polymerase X Enables Rapid Detection of Sequence-Specific DNA Targets

Abstract

Mimiviral polymerase X, mvPolX, is a repair polymerase that is involved in base excision repair (BER) and carries out the gap-filling function in double-stranded DNA (dsDNA). We demonstrate a sensitive and sequence-specific DNA detection method using this polymerase. mvPolX begins polymerizing DNA from the 3 end of a gap, displacing the downstream nucleotides without exonuclease activity. Our detection method is built on this activity of mvPolX. We designed a probe molecule consisting of a partial dsDNA with a 3 over-hang region complementary to the target DNA to be detected. The probe has a fluorophore-quencher (FAM-BHQ1) tag to facilitate detection upon strand removal. Binding of the probe to the complementary target forms a dsDNA with a single nucleotide gap in one strand. mvPolX binds this gap region and begins polymerisation eventually displacing the quencher strand leading to an increase in fluorescence. Proof-of-concept has been established using a synthetic 19 bp target DNA sequence. The method is specific and did not show any strand displacement when a single or double mismatched nucleotide at the 3 end of the target DNA was used. To demonstrate this molecular assay, we used M13 phage as our target. Asymmetric PCR (aPCR) was used to obtain single-stranded target DNA (158 bases) from M13 genomic DNA, which was directly used in the assay as target. The combination of aPCR and mvPolX assay can detect as low as 10 copies of genomic DNA. The enzymatic reaction is fast, requiring only 15 min of incubation with mvPolX at 30 {degrees}C. We have further demonstrated the efficiency of the assay in presence of multiple non-target DNA by detecting the target DNA from M13 phage spiked lakewater samples.