Single Base Specificity
Edited and unedited gene sequences are detected using padlock probe chemistry.
Padlock probes are linear oligonucleotides that are designed to target either edited or wildtype gene sequences. Only in the event of a perfect sequence match, a high-fidelity ligase is able to join together the ends of the probe, this process “locks” the probe on its target. This powerful combination enables differentiation of down to single base mismatches.
PCR-independent Signal Amplification
Individual reacted probes are copied using rolling circle amplification.
To visualize individual reacted padlock probes, a DNA polymerase with high processivity and strand displacement properties copies the original probe sequence hundreds of times. This produces individual DNA molecules of sub-micron size that are encoded with original probe sequences that can include specific detection tag sequences.
Counting Individual Gene Edited Events
Amplified probes are quantified by fluorescence microscopy.
Individual amplified DNA molecules are labeled with fluorescently tagged oligonucleotides. As a result, locked and amplified probes are visualized under fluorescence microscopy as individual bright spots with excellent signal-to-noise ratio. Discrete fluorescence signals correspond to individual edited or unedited events. By counting the number of resulting spots the efficiency of a gene editing process can be determined with high precision.
Highly Efficient Single Molecule Counting
Labeled molecules are pulled and captured into the area of a single field of view of a low magnification microscope objective. Enabling highly efficient imaging acquisition and fast computational image analysis.