QueSTR probes: Quencher-labeled RNase H2-dependent probes for Short Tandem Repeat genotyping

Forensic Short Tandem Repeat (STR) genotyping is almost exclusively performed by capillary electrophoresis (CE) in specialized laboratories. As an alternative to CE, and to enable miniaturized lab-on-a-chip STR profiling, we developed the QueSTR probes, a hybridization-based genotyping assay that relies on the recognition and cleavage of an RNA:DNA duplex by the RNase H2 enzyme. For each STR allele to be genotyped, a matching DNA probe containing one RNA moiety is designed. After performing asymmetric STR PCR, a hybridization curve analysis indicates the matching probe(s), and thus indicates the allele(s) present in the sample. Accurate genotyping of 13 samples was obtained using the QueSTR probes for three CODIS core loci (D16S539, D7S820, and TH01). A probe corresponding to the TH01 9.3 allele was included to demonstrate accurate genotyping, even in the presence of a partial repeat. The QueSTR probes are a valuable option to miniaturize STR genotyping in lab-on-a-chip devices that cannot harbor a CE analysis. •Novel STR genotyping probes developed.•Probes are cleaved by the RNase H2 enzyme upon hybridization.•Successful genotyping obtained, even in presence of partial repeats.