Ultrafast enzymatic digestion of deoxyribonucleic acid in aqueous microdroplets for sequence discrimination and identification.

We report the use of aqueous microdroplets to accelerate deoxyribonucleic acid (DNA) fragmentation by deoxyribonuclease I (DNase I), and we present a simple, ultrafast approach named DNA fragment mass fingerprinting to discriminate different DNA sequences by comparing their fragment mass patterns. DNA fragmentation in tiny microdroplets, which was produced by electrosonically spraying (+3 kV) a room temperature aqueous solution containing 10 μM DNA and 10 μg ml^-1 DNase I from a homemade setup, takes less than 1 ms. High differentiation/identification fidelity could be obtained by applying a cosine correlation measure for similarity assessment between two fragment mass patterns, which compares both mass-to-charge ratios (m/z) with an error tolerance of 5 ppm and the peaks' relative intensities. A single-nucleotide mutation in the sequence of bases, as exemplified by the sickle cell anemia mutation, is differentiated by setting a cutoff value of similarity at 90%. The order change of two adjacent bases in the sequence could still be well discriminated with a similarity of only 62% between the fragment mass patterns of the two similar sequences, which have the same molecular weights and thus cannot be differentiated by gel electrophoresis or direct mass detection by mass spectrometry. Compared to traditional genotyping methods, such as quantitative real-time polymerase chain reaction, the identification process with our approach could be completed within several minutes without any other expensive and complicated reagents or experimental steps. The potential of our approach for convenient and fast microbe genetic discrimination or identification is further demonstrated by differentiating the Orf1ab gene fragments of two similar coronaviruses with a very high sequence homologous rate of 96%, SARS-CoV-2 and bat-SL-CoVZC45, with a similarity of 0% between their fragment mass patterns.