3′ Tth Endonuclease Cleavage Polymerase Chain Reaction (3TEC-PCR) Technology for Single-Base-Specific Multiplex Pathogen Detection using a Two-Oligonucleotide System

Polymerase chain reaction (PCR) is the standard in nucleic acid amplification technology for infectious disease pathogen detection and has been the primary diagnostic tool employed during the global COVID-19 pandemic. Various PCR technology adaptations, typically using two-oligonucleotide dye-binding methods or three-oligonucleotide hydrolysis probe systems, enable real-time multiplex target detection or single-base specificity for the identification of single-nucleotide polymorphisms (SNPs). A small number of two-oligonucleotide PCR systems facilitating both multiplex detection and SNP identification have been reported; however, these methods often have limitations in terms of target specificity, production of variable or false-positive results, and the requirement for extensive optimisation or post-amplification analysis. This study introduces 3′ Tth endonuclease cleavage PCR (3TEC-PCR), a two-oligonucleotide PCR system incorporating a modified primer/probe and a thermostable cleavage enzyme, Tth endonuclease IV, for real-time multiplex detection and SNP identification. Complete analytical specificity, low limits of detection, single-base specificity, and simultaneous multiple target detection have been demonstrated in this study using 3TEC-PCR to identify bacterial meningitis associated pathogens. This is the first report of a two-oligonucleotide, real-time multiplex PCR technology with single-base specificity using Tth endonuclease IV.

CRISPR/Cas9-Based Lateral Flow and Fluorescence Diagnostics

Clustered regularly interspaced short palindromic repeat (CRISPR/Cas) proteins can be designed to bind specified DNA and RNA sequences and hold great promise for the accurate detection of nucleic acids for diagnostics. We integrated commercially available reagents into a CRISPR/Cas9-based lateral flow assay that can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequences with single-base specificity. This approach requires minimal equipment and represents a simplified platform for field-based deployment. We also developed a rapid, multiplex fluorescence CRISPR/Cas9 nuclease cleavage assay capable of detecting and differentiating SARS-CoV-2, influenza A and B, and respiratory syncytial virus in a single reaction. Our findings provide proof-of-principle for CRISPR/Cas9 point-of-care diagnosis as well as a scalable fluorescent platform for identifying respiratory viral pathogens with overlapping symptomology.

A CRISPR-Cas autocatalysis-driven feedback amplification network for supersensitive DNA diagnostics

Artificial nucleic acid circuits with precisely controllable dynamic and function have shown great promise in biosensing, but their utility in molecular diagnostics is still restrained by the inability to process genomic DNA directly and moderate sensitivity. To address this limitation, we present a CRISPR-Cas–powered catalytic nucleic acid circuit, namely, CRISPR-Cas–only amplification network (CONAN), for isothermally amplified detection of genomic DNA. By integrating the stringent target recognition, helicase activity, and trans-cleavage activity of Cas12a, a Cas12a autocatalysis-driven artificial reaction network is programmed to construct a positive feedback circuit with exponential dynamic in CONAN. Consequently, CONAN achieves one-enzyme, one-step, real-time detection of genomic DNA with attomolar sensitivity. Moreover, CONAN increases the intrinsic single-base specificity of Cas12a, and enables the effective detection of hepatitis B virus infection and human bladder cancer–associated single-nucleotide mutation in clinical samples, highlighting its potential as a powerful tool for disease diagnostics.

Cascade CRISPR/cas enables amplification-free microRNA sensing with fM-sensitivity and single-base-specificity

A cascade CRISPR/cas nucleic acid diagnostic system, which can achieve high-sensitive and single-base specificity without target amplification, was developed for miRNA detection.

TruSight Oncology 500: Enabling Comprehensive Genomic Profiling and Biomarker Reporting with Targeted Sequencing

BackgroundAs knowledge of mechanisms that drive the development of cancer grows, there has been corresponding growth in therapies specific to a mechanism. While these therapies show improvements in patient outcomes, they can be expensive and are effective only for a subset of patients. These treatments drive interest in research focused on the assignment of cancer therapies based on aberrations in individual genes or biomarkers that assess the broader mutational landscape, including microsatellite instability (MSI) and tumor mutational burden (TMB).MethodsHere we describe the TruSight™ Oncology 500 (TSO500; Research Use Only) bioinformatics workflow. This tumor-only approach leverages the next-generation sequencing-based assay TSO500 to enable high fidelity determination of DNA variants across 523 cancer-relevant genes, as well as MSI status and TMB in formalin-fixed paraffin-embedded (FFPE) samples.ResultsThe TSO500 bioinformatic workflow integrates unique molecular identifier (UMI)-based error correction and a dual approach variant filtering strategy that combines statistical modeling of error rates and database annotations to achieve detection of variants with allele frequency approaching 5% with 99.9998% per base specificity and 99% sensitivity in FFPE samples representing a variety of tumor types. TMB determined using the tumor-only workflow of TSO500 correlated well with tumor-normal (N =170, adjusted R2=0.9945) and whole-exome sequencing (N=108, adjusted R2=0.933). Similarly, MSI status determined by TSO500 showed agreement (N=106, 98% agreement) with a MSI-PCR assay.ConclusionTSO500 is an accurate tumor-only workflow that enables researchers to systematically characterize tumors and identify the next generation of clinical biomarkers.

VERBALIZATION OF THE"FATE" CONCEPT IN THE CHINESE LINGUISTIC WORLDVIEW

Purpose of the study: The article has the following objectives: to describe the specificity of the axiological concept of "fate" in Chinese; to determine coordinates (cognitive base, specificity, location and type) of the concept of "fate" on the Chinese conceptual maps; to identify the main lexical markers of the concept of "fate" in the scientific and popular-poetic discourse; to define classifiers and to determine the place of the concept of "fate" among related concepts. Methodology: The authors employed the following methods of conceptual analysis and cognitive modeling: diachronic and synchronic methods in combination with the comparative method. The results of the research contribute to the linguistic mapping of the concept of "fate" and related concepts in the Chinese and Kazakh languages. Main Findings: This article contributes to conceptology as an area of linguistics in general and explores the linguistic specificity of the axiological concept of "fate" in Chinese as an important marker of national mentality. Applications of this study: The future studies of the concept of "fate" and its related concepts should involve genetically close and distant languages, in different types of discourse. It is interesting and relevant to further examine its connections with other concepts, such as "life", "death", "happiness", "love", "soul", "way", etc. The continued research in this direction has important practical significance for translation and cultural studies. Novelty/Originality of this study: By analyzing the content of linguistic signs of the concept of "fate" one can describe the framework of human worldview, determine its connection with the true world, passing it through the "filter of knowledge", and determine its place in the conceptual picture of the universe. If its philosophical cognitive essence was misunderstood, it could not become a concentrate of other worldwide concepts, the universal true being.

Novel Assay for Quantitative Analysis of DNA Methylation at Single-Base Resolution

BACKGROUND The DNA methylation profile provides valuable biological information with potential clinical utility. Several methods, such as quantitative methylation-specific PCR (qMSP), have been developed to examine methylation of specific CpG sites. Existing qMSP-based techniques fail to examine the genomic methylation at a single-base resolution, particularly for loci in gene bodies or extensive CpG open seas lacking flanking CpGs. Therefore, we established a novel assay for quantitative analysis of single-base methylation. METHODS To achieve a robust single-base specificity, we developed a PCR-based method using paired probes following bisulfite treatment. The 6-carboxyfluorescein- and 2′-chloro-7′phenyl-1,4-dichloro-6-carboxy-fluorescein-labeled probes conjugated with minor groove binder were designed to specifically bind to the methylated and unmethylated allele of targeted single CpGs at their 3′ half regions, respectively. The methylation percentage was calculated by values of methylation / (methylation + unmethylation). RESULTS In the detection of single CpGs within promoters or bodies of 4 human genes, the quantitative analysis of the single-base methylation assay showed a detection capability in the 1 to 1:10000 dilution experiments with linearity over 4 orders of magnitude (R2 = 0.989–0.994; all P < 0.001). In a cohort of 10 colorectal cancer samples, the assay showed a comparable detection performance with bisulfite pyrosequencing (R2 = 0.875–0.990; all P < 0.001), which was better than conventional qMSP methods normalized by input control reaction (R2 = 0.841 vs 0.769; P = 0.002 vs 0.009). CONCLUSIONS This assay is highly specific and sensitive for determining single-base methylation and, thus, is potentially useful for methylation-based panels in diagnostic and prognostic applications.

The crystal structure of dGTPase reveals the molecular basis of dGTP selectivity

Deoxynucleotide triphosphohydrolases (dNTPases) play a critical role in cellular survival and DNA replication through the proper maintenance of cellular dNTP pools. While the vast majority of these enzymes display broad activity toward canonical dNTPs, such as the dNTPase SAMHD1 that blocks reverse transcription of retroviruses in macrophages by maintaining dNTP pools at low levels, Escherichia coli (Ec)-dGTPase is the only known enzyme that specifically hydrolyzes dGTP. However, the mechanism behind dGTP selectivity is unclear. Here we present the free-, ligand (dGTP)- and inhibitor (GTP)-bound structures of hexameric Ec-dGTPase, including an X-ray free-electron laser structure of the free Ec-dGTPase enzyme to 3.2 Å. To obtain this structure, we developed a method that applied UV-fluorescence microscopy, video analysis, and highly automated goniometer-based instrumentation to map and rapidly position individual crystals randomly located on fixed target holders, resulting in the highest indexing rates observed for a serial femtosecond crystallography experiment. Our structures show a highly dynamic active site where conformational changes are coupled to substrate (dGTP), but not inhibitor binding, since GTP locks dGTPase in its apo- form. Moreover, despite no sequence homology, Ec-dGTPase and SAMHD1 share similar active-site and HD motif architectures; however, Ec-dGTPase residues at the end of the substrate-binding pocket mimic Watson–Crick interactions providing guanine base specificity, while a 7-Å cleft separates SAMHD1 residues from dNTP bases, abolishing nucleotide-type discrimination. Furthermore, the structures shed light on the mechanism by which long distance binding (25 Å) of single-stranded DNA in an allosteric site primes the active site by conformationally “opening” a tyrosine gate allowing enhanced substrate binding.