A single-molecule counting approach for convenient and ultrasensitive measurement of restriction digest efficiencies.

Restriction endonucleases play a central role in the microbial immune system against viruses and are widely used in DNA specific cleavage, which is called restriction digestion, for genetic engineering. Herein, we applied digital cell-free protein synthesis as an easy-to-use orthogonal readout means to assess the restriction digest efficiency, a new application of digital bioassays. The digital counting principle enabled an unprecedentedly sensitive trace analysis of undigested DNA at the single-molecule level in a PCR-free manner. Our approach can quantify the template DNA of much lower concentrations that cannot be detected by ensemble-based methods such as gold-standard DNA electrophoresis techniques. The sensitive and quantitative measurements revealed a considerable variation in the digest efficiency among restriction endonucleases, from less than 70% to more than 99%. Intriguingly, none of them showed truly complete digestion within reasonably long periods of reaction time. The same rationale was extended to a multiplexed assay and applicable to any DNA-degrading or genome-editing enzymes. The enzyme kinetic parameters and the flanking sequence-dependent digest efficiency can also be interrogated with the proposed digital counting method. The absolute number of residual intact DNA molecules per microliter was concluded to be at least 107, drawing attention to the residual issue of genetic materials associated with the interpretation of nucleases' behaviors and functions in daily genetic engineering experiments.