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

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.

SUBMITTER: Zhong X

PROVIDER: S-EPMC8185430 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Authentic identification of plants is essential for exploiting their medicinal properties as well as to stop the adulteration and malpractices with the trade of the same. OBJECTIVE:To identify a herbal powder obtained from a herbalist in the local vicinity of Rajkot, Gujarat, using deoxyribonucleic acid (DNA) barcoding and molecular tools. MATERIALS AND METHODS:The DNA was extracted from a herbal powder and selected Cassia species, followed by the polymerase chain reaction (PCR) and sequencing of the rbcL barcode locus. Thereafter the sequences were subjected to National Center for Biotechnology Information (NCBI) basic local alignment search tool (BLAST) analysis, followed by the protein three-dimension structure determination of the rbcL protein from the herbal powder and Cassia species namely Cassia fistula, Cassia tora and Cassia javanica (sequences obtained in the present study), Cassia Roxburghii, and Cassia abbreviata (sequences retrieved from Genbank). Further, the multiple and pairwise structural alignment were carried out in order to identify the herbal powder. RESULTS:The nucleotide sequences obtained from the selected species of Cassia were submitted to Genbank (Accession No. JX141397, JX141405, JX141420). The NCBI BLAST analysis of the rbcL protein from the herbal powder showed an equal sequence similarity (with reference to different parameters like E value, maximum identity, total score, query coverage) to C. javanica and C. roxburghii. In order to solve the ambiguities of the BLAST result, a protein structural approach was implemented. The protein homology models obtained in the present study were submitted to the protein model database (PM0079748-PM0079753). The pairwise structural alignment of the herbal powder (as template) and C. javanica and C. roxburghii (as targets individually) revealed a close similarity of the herbal powder with C. javanica. CONCLUSION:A strategy as used here, incorporating the integrated use of DNA barcoding and protein structural analyses could be adopted, as a novel rapid and economic procedure, especially in cases when protein coding loci are considered. SUMMARY:Authentic identification of plants is essential for exploiting their medicinal properties as well as to stop the adulteration and malpractices with the trade of the same. A herbal powder was obtained from a herbalist in the local vicinity of Rajkot, Gujarat. An integrated approach using DNA barcoding and structural analyses was carried out to identify the herbal powder. The herbal powder was identified as Cassia javanica L.

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

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