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

Xiaoqin Zhong,Richard N Zare,Hao Chen

doi:10.1017/qrd.2021.2

Abstract

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.

Highlights

In the past few years, it has been demonstrated that reactions run in microdroplets can be remarkably accelerated compared to the same reactions in bulk solvents (Wei et al, 2020)
Microdroplets containing 10-μM deoxyribonucleic acid (DNA) sample and 10-μg mlÀ1 enzyme of deoxyribonuclease I (DNase I) dissolved in 5-mM buffer of ammonium acetate were generated by a homemade electrosonic sprayer in which a sheath of rapidly flowing dry N2 gas at 120 psi surrounded an inner capillary held at typically +3 kV
For aqueous droplets with an initial size of 9 μm produced by electrosonic spray ionisation (ESSI) in our case, the time required for the first droplet fission to occur is longer than 547 μs owing to the lower vapour pressure of water compared to heptane

Summary

Introduction

In the past few years, it has been demonstrated that reactions run in microdroplets can be remarkably accelerated compared to the same reactions in bulk solvents (Wei et al, 2020). Polymerase chain reaction (PCR) is widely used to amplify and determine a chosen genomic region by designing a PCR primer and a probe labelled with a flaring group to report the existence of the chosen genomic region or not, which complicates the genotyping process and is less cost-efficient (Van Elden et al, 2001; Gibson, 2006; Baker, 2010) In contrast to these traditional methods mentioned above, most of which take several hours and require expensive and complicated reagents, we present an alternative approach that makes only use of a DNA exonuclease to digest DNA samples in aqueous microdroplets within microseconds. When the microdroplets are analysed with a high-resolution mass spectrometer, this procedure normally takes several minutes and differentiates two fragments with a mass shift larger than 5 ppm

Methods

Results

Conclusion