Detection Of Single-base Mutations Research Articles

An enzyme-free fluorescence sensing platform based on multiplex toehold-mediated strand displacement for point-of-care testing of single nucleotide polymorphisms

Single nucleotide polymorphisms (SNPs) are biomarkers for the prediction and diagnosis of biomedical diseases, as well as important reference markers for intelligent design and breeding of animals and plants. Therefore, the development of high-sensitivity, practicability, and convenient SNPs sensing strategies and detection platforms is of great significance. In this study, we pioneered an ultra-sensitive universal sensing strategy for single base mutation detection and an enzyme-free FRET fluorescence sensing point-of-care testing (POCT) platform for SNP detection. Based on the thermodynamically regulated strand replacement mechanism, the system rationally introduced extra base mismatches and combined multiple toehold-mediated strand displacement (TMSD) reactions to achieve enzyme-free recognition of the single base mutation and double amplification of the detection signal. Finally, the biological signal of the mutant target (MT) was recognized, amplified, and transduced into a fluorescence resonance energy transfer (FRET) signal between Cy3 and Cy5 for output. Overall, the proposed biosensing strategy demonstrated high sensitivity for MT, with a detection limit as low as 0.37 fM, and could achieve detection of targets with mutant abundances as low as 0.1 %. Moreover, a portable detection system was assembled in conjunction with this sensing strategy to form a POCT platform for SNP genotyping. This POCT platform was validated to accurately identify SNPs from human cancer cells and soybean leaf genome extracts. In summary, this study will provide new insights into the design of enzyme-free biosensors for SNP detection, and will provide important technical support tools for the prevention and diagnosis of major diseases, animal and plant breeding, etc.

Double base mismatches mediated catalytic hairpin assembly for enzyme-free single-base mutation detection: integrating signal recognition and amplification in one.

Single nucleotide polymorphism (SNP) biosensors are emerging rapidly for their promising applications in human disease prevention diagnosis, treatment, and prognosis. However, it remains a bottleneck in equipping simple and stable biosensors with the traits of high sensitivity, non-enzyme, and low cost. Double base mismatches mediated chain displacement reactions have attracted fascinating advantages of tailorable thermodynamics stability, non-enzyme, and excellent assembly compliance to involvement in SNP identification. As the base mismatch position and amount in DNA sequence can be artificially adjusted, it provides plenty of selectivity and specificity for exploring perfect biosensors. Herein, abiosensor with double base mismatches mediated catalytic hairpin assembly (CHA) is designed via one base mismatch in the toehold domain and the other base mismatch in the stem sequence of hairpin 1 (H1) by triggering CHA reaction to achieve selective amplification of the mutation target (MT) and fluorescence resonance energy transfer (FRET) effect that is composed of Cy3 and Cy5 terminally attached H1 and hairpin 2 (H2). Depending on the rationally designed base mismatch position and toehold length, the fabricated biosensors show superior SNP detection performance, exhibiting a good linearity with high sensitivity of 6.6 fM detection limitand a broad detection abundance of 1%. The proposed biosensor can be used to detect the KRAS mutation gene in real samples and obtain good recoveries between 106 and 116.99%. Remarkably, these extendible designs of base mismatches can be used for more types of SNP detection, providing flexible adjustment based on base mismatch position and toehold length variations, especially for their thermodynamic model for DNA-strand displacement reactions.

CRISPR-Mediated Profiling of Viral RNA at Single-Nucleotide Resolution.

Mass pathogen screening is critical to preventing the outbreaks and spread of infectious diseases. The large-scale epidemic of COVID-19 and the rapid mutation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus have put forward new requirements for virus detection and identification techniques. Here, we report a CRISPR-based Amplification-free Viral RNA Electrical Detection platform (CAVRED) for the rapid detection and identification of SARS-CoV-2 variants. A series of CRISPR RNA assays were designed to amplify the CRISPR-Cas system's ability to discriminate between mutant and wild RNA genomes with a single-nucleotide difference. The identified viral RNA information was converted into readable electrical signals through field-effect transistor biosensors for the achievement of highly sensitive detection of single-base mutations. CAVRED can detect the SARS-CoV-2 virus genome as low as 1 cp μL-1 within 20 mins without amplification, and this value is comparable to the detection limit of real-time quantitative polymerase chain reaction. Based on the excellent RNA mutation detection ability, an 8-in-1 CAVRED array was constructed and realized the rapid identification of 40 simulated throat swab samples of SARS-CoV-2 variants with a 95.0 % accuracy. The advantages of accuracy, sensitivity, and fast speed of CAVRED promise its application in rapid and large-scale epidemic screening.

Sequence terminus dependent PCR for site-specific mutation and modification detection

The detection of changes in nucleic acid sequences at specific sites remains a critical challenge in epigenetics, diagnostics and therapeutics. To date, such assays often require extensive time, expertise and infrastructure for their implementation, limiting their application in clinical settings. Here we demonstrate a generalizable method, named Specific Terminal Mediated Polymerase Chain Reaction (STEM-PCR) for the detection of DNA modifications at specific sites, in a similar way as DNA sequencing techniques, but using simple and widely accessible PCR-based workflows. We apply the technique to both for site-specific methylation and co-methylation analysis, importantly using a bisulfite-free process - so providing an ease of sample processing coupled with a sensitivity 20-fold better than current gold-standard techniques. To demonstrate the clinical applicability through the detection of single base mutations with high sensitivity and no-cross reaction with the wild-type background, we show the bisulfite-free detection of SEPTIN9 and SFRP2 gene methylation in patients (as key biomarkers in the prognosis and diagnosis of tumours).

Two CRISPR/Cas12a-based methods for fast and accurate detection of single-base mutations.

Current single-base mutation detection approaches are time-consuming, labor-intensive, and costly. This highlights the critical need for speedy and accurate technology capable of detecting single-base alterations. Using clustered regularly interspaced short palindromic repeats/associated protein 12a (CRISPR/Cas12a), two fundamental approaches for getting 100% differentiation of single-base mutations have been established, by which fluorescence signals could be detected for variants but not for wild strains. The first method required both polymerase chain reaction (PCR) and CRISPR/Cas12a cleavage: By introducing a mismatched base at the 3' end of the primers and adjusting the PCR settings, the wild strain strand amplifications were completely blocked prior to CRISPR/Cas12a cleavage. The parameters for Method 1 (PCR+CRISPR/Cas12a) could be easily controlled and adjusted to attain a sensitivity of one copy (about 6 copies μL-1). The second method included isothermal recombinase polymerase amplification (RPA) and CRISPR/Cas12a cleavage: By introducing an extra mismatched base adjacent to the single-base mutant site by RPA (IMAS-RPA), the RPA products from the wild strains were rendered incapable of triggering the cleavage activity of CRISPR/Cas12a. Method 2 (IMAS-RPA) was rapid and easy to implement (can be finished within 1h). Because each method has its own set of advantages, the laboratory environment-appropriate methods can be selected independently. Both approaches are expected to aid in clinical diagnosis to some extent in the near future.

Interference reduction isothermal nucleic acid amplification strategy for COVID-19 variant detection

Common reference methods for COVID-19 variant diagnosis include viral sequencing and PCR-based methods. However, sequencing is tedious, expensive, and time-consuming, while PCR-based methods have high risk of insensitive detection in variant-prone regions and are susceptible to potential background signal interference in biological samples. Here, we report a loop-mediated interference reduction isothermal nucleic acid amplification (LM-IR-INA) strategy for highly sensitive single-base mutation detection in viral variants. This strategy exploits the advantages of nicking endonuclease-mediated isothermal amplification, luminescent iridium(III) probes, and time-resolved emission spectroscopy (TRES). Using the LM-IR-INA strategy, we established a luminescence platform for diagnosing COVID-19 D796Y single-base substitution detection with a detection limit of 2.01 × 105 copies/μL in a linear range of 6.01 × 105 to 3.76 × 108 copies/μL and an excellent specificity with a variant/wild-type ratio of significantly less than 0.0625%. The developed TRES-based method was also successfully applied to detect D796Y single-base substitution sequence in complicated biological samples, including throat and blood, and was a superior to steady-state technique. LM-IR-INA was also demonstrated for detecting the single-base substitution D614G as well as the multiple-base mutation H69/V70del without mutual interference, indicating that this approach has the potential to be used as a universal viral variant detection strategy.

CRISPR/Cas9 mediated triple signal amplification platform for high selective and sensitive detection of single base mutations

Single base mutations detection is crucial for the diagnosis and treatment of cancer. However, the current methods with poor selectivity and sensitivity required large instruments, which are difficult to meet clinical demands. Herein, we develop a CRISPR/Cas9 based visual colorimetric platform to specifically detect all single base mutations. In this strategy, the Recombinase Polymerase Amplification (RPA) was firstly used to amplify the target, and introduced the PAM site in the target DNA sequence by designing the point mutation primer, thus achieving detection for all single base mutations by the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated specific recognition. With the help of CRISPR/Cas9 system, those RPA products can release single strand DNA to hybridize with the padlock probe and trigger rolling circle amplification (RCA). Based on the magnetic separation, HRP-gold nanoparticles complex (hGNPs) and biotin modified probe (Bio-probe) were further used to achieve enhanced visual variations assay by hybridizing with RCA products. Benefiting from the RPA assisted triple signal amplification, this method not only showed enhanced sensitivity with a limit of detection (LOD) as low as 0.2 fM and 0.01% of KRAS-G12D mutation percentage, but the specificity against KRAS-G12D mutation also be synergistically enhanced by combining the CRISPR/Cas9-mediated specific recognition with the specific T4 ligation reaction of RCA system. Furthermore, this system has been successfully used to visually detect genome in serum, suggesting its great potential for point-of-care diagnosis in clinical.

A universal probe system for low-abundance point mutation detection based on endonuclease IV.

Single base mutations are closely related to cancer diagnosis and treatment. The fluorescent probe method is one of the important methods to detect single-base mutations. We constructed a universal probe detection system based on endonuclease IV and the DNA strand displacement reaction. The system uses two toehold strand displacement reactions to relay the mutation information to the universal strand. There is no need to design the probe one-by-one for each mutation point during multi-site detection. It has the advantages of simple operation, rapid detection, and low cost. We used this method to detect common clinical mutation sites (PTEN R130Q/EGFR L858R/PTEN rs1473918395), and the detection limit can reach 0.1%-1%. The detection system can provide a new rapid and economical method for clinical single-base mutation detection, and has broad application prospects in diagnosis and prognostic evaluation.

Demonstration of a Superior Deep-UV Surface-Enhanced Resonance Raman Scattering (SERRS) Substrate and Single-Base Mutation Detection in Oligonucleotides.

In life science, rapid mutation detection in oligonucleotides is in a great demand for genomic and medical screening. To satisfy this demand, surface-enhanced resonance Raman spectroscopy (SERRS) in the deep-UV (DUV) regime offers a promising solution due to its merits of label-free nature, strong electromagnetic confinement, and charge transfer effect. Here, we demonstrate an epitaxial aluminum (Al) DUV-SERRS substrate that resonates effectively with the incident Raman laser and the ss-DNA at 266 nm, yielding significant SERRS signals of the detected analytes. For the first time, to the best of our knowledge, we obtaine SERRS spectra for all bases of oligonucleotides, not only revealing maximum characteristic Raman peaks but also recording the highest enhancement factor of up to 106 for a 1 nm thick adenine monomer. Moreover, our epitaxial Al DUV-SERRS substrate is able to enhance the Raman signal of all four bases of 12-mer ss-DNA and to further linearly quantify the single-base mutation in the 12-mer ss-DNA.

Taqman-MGB nanoPCR for Highly Specific Detection of Single-Base Mutations.

PurposeDetection of single-base mutations is important for real-time monitoring of tumor progression, therapeutic effects, and drug resistance. However, the specific detection of single-base mutations from excessive wild-type background sequences with routine PCR technology remains challenging. Our objective is to develop a simple and highly specific qPCR-based single-base mutation detection method.MethodsUsing EGRF T790M as a model, gold nanoparticles at different concentrations were separately added into the Taqman-MGB qPCR system to test specificity improvement, leading to the development of the optimal Taqman-MGB nanoPCR system. Then, these optimal conditions were used to test the range of improvement in the specificity of mutant-type and wild-type templates and the detection limit of mutation abundances in a spiked sample.ResultsThe Taqman-MGB nanoPCR was established based on the traditional qPCR, with significantly suppressed background noise and improved specificity for single-base mutation detection. With EGFR T790M as a template, we demonstrated that our Taqman-MGB nanoPCR system could improve specificity across a wide concentration range from 10−9 μM to 10 μM and detect as low as 0.95% mutation abundance in spiked samples, which is lower than what the traditional Taqman-MGB qPCR and existing PCR methods can detect. Moreover, we also proposed an experimentally validated barrier hypothesis for the mechanism of improved specificity.ConclusionThe developed Taqman-MGB nanoPCR system could be a powerful tool for clinical single-base mutation detection.

Ultrasensitive Detection of RNA with Single-Base Resolution by Coupling Electrochemical Sensing Strategy with Chimeric DNA Probe-Aided Ligase Chain Reaction.

Accurate and sensitive detection of single-base mutations in RNAs is of great value in basic studies of life science and medical diagnostics. However, the current available RNA detection methods are challenged by heterogeneous clinical samples in which trace RNA mutants usually existed in a large pool of normal wild sequences. Thus, there is still great need for developing the highly sensitive and highly specific methods in detecting single-base mutations of RNAs in heterogeneous clinical samples. In the present study, a new chimeric DNA probe-aided ligase chain reaction-based electrochemical method (cmDNA-eLCR) was developed for RNA mutation detection through the BSA-based carrier platform and the horseradish peroxidase-hydrogen peroxide-tetramethylbenzidine (HRP-H2O2-TMB) system. The denaturing polyacrylamide gel electrophoresis and a fluorophore-labeled probe was ingeniously designed to demonstrate the advantage of cmDNA in ligation to normal DNA templated by RNA with the catalysis of T4 RNA ligase 2 as well as its higher selectivity than DNA ligase system. Finally, the proposed cmDNA-eLCR, compared with the traditional eLCR, showed excellent performance in discriminating single base-mismatched sequences, where the signal response for mismatched targets at a high concentration could overlap completely with that for the blank control. Besides, this cmDNA-eLCR assay had a wide linear range crossing six orders of magnitude from 1.0 × 10-15 to1.0 × 10-10 M with a limit of detection as low as 0.6 fM. Furthermore, this assay was applied to detect RNA in real sample with a satisfactory result, thereby demonstrating its great potential in diagnosis of RNA-related diseases.

Detection of single-base mutation of DNA oligonucleotides with different lengths by terahertz attenuated total reflection microfluidic cell

Many human genetic diseases are caused by single-base mutation in the gene sequence. Since DNA molecules with single-base mutation are extremely difficult to differentiate, existing detection methods are invariably complex and time-consuming. We propose a new label-free and fast terahertz (THz) spectroscopic technique based on a home-made terahertz attenuated total reflection (ATR) microfluidic cell and a terahertz time-domain spectroscopy (THz-TDS) system to detect single-base-mutated DNA molecules. The detected object DNA molecules are normal hemoglobin gene, sickle cell anemia gene (15 nt), JAK2 gene wild type and JAK2 V617F gene mutation (39 nt) from sickle cell anemia and thrombocytopenia, respectively. Results show that the oligonucleotide fragments with single-base mutation can be identified by THz spectroscopy combined with the ATR microfluidic cell, and the recognition effect of short oligonucleotide fragments with single-base mutation is better than that of long oligonucleotide fragments. The terahertz biosensor is shown to have high sensitivity and can be used to detect DNA molecules directly in the solution environment.

Determination of low-abundance single-base point mutations based on endonuclease IV and branch migration system

Combining endonuclease IV and branch migration competition systems, we have designed a new single-base mutation detection method which is capable of thermostatic, sensitive, simple, and cost-effective at the same time, while other probe method based on endonuclease IV hardly achieve. Our method has better discrimination factors (6.81–83.10) and a low abundance detection limit of mutant-type DNA (MT) (0.05% mutant-type DNA/total DNA) without complex temperature optimize process. The concentration detection limit of MT is 0.03 nM.The abundance detection limit for EGFR L858R (0.1%) and PTEN R130Q (0.05%) in clinical samples suggested that the method has potential applications in clinical diagnosis.

Redefining Molecular Amphipathicity in Reversing the "Coffee-Ring Effect": Implications for Single Base Mutation Detection.

The "coffee ring effect" is a natural phenomenon wherein sessile drops leave ring-shaped structures on the solid surfaces upon drying. It drives a nonuniform deposition of suspended compounds on the substrates, which adversely affects many processes, including surface-assisted biosensing and molecular self-assembly. In this study, we describe how the coffee ring effect can be eliminated by controlling the amphipathicity of the suspended compounds, for example, DNA modified with hydrophobic dye. Specifically, nuclease digestion of the hydrophilic DNA end converts the dye-labeled molecule into an amphipathic molecule (one with comparably weighted hydrophobic and hydrophilic ends) and reverses the coffee ring effect and results in a uniform disk-shaped feature deposition of the dye. The amphipathic product decreases the surface tension of the sessile drops and induces the Marangoni flow, which drives the uniform distribution of the amphipathic dye-labeled product in the drops. As a proof of concept, this strategy was used in a novel enzymatic amplification method for biosensing to eliminate the coffee ring effect on a nitrocellulose membrane and increase assay reliability and sensitivity. Importantly, the reported strategy for eliminating the coffee ring effect can be extended to other sessile drop systems for potentially improving assay reliability and sensitivity.

Rapid KRAS Mutation Detection via Hybridization-Induced Aggregation of Microbeads.

Using hybridization-induced aggregation (HIA), a unique bead-based DNA detection technology scalable for a microchip platform, we describe a simplistic, low-cost method for rapid mutation testing. HIA utilizes a pair of sequence-specific oligonucleotide probes bound to magnetic microbeads. Hybridization to a target DNA strand tethers the beads together, inducing bead aggregation. By simply using the extent of bead aggregation as a measure of the hybridization efficiency, we avoid the need for additional labels and sophisticated analytical equipment. Through strategic manipulation of the assay design and experimental parameters, we use HIA to facilitate, for the first time, the detection of single base mutations in a gene segment and, specifically, the detection of activating KRAS mutations. Following the development and optimization of the assay, we apply it for KRAS mutation analysis of four human cancer cell lines. Ultimately, we present a proof-of-principle method for detecting any of the common KRAS mutations in a single-step, 2 min assay, using only one set of oligonucleotide probes, for a total analysis time of less than 10 min post-PCR. The assay is performed at room temperature and uses simple, inexpensive instrumentation that permits multiplexed analysis.

Detection of BRAF mutations from solid tumors using Tumorplex™ technology

Allele specific multiplex sequencing (Tumorplex™) is a new molecular platform for the detection of single base mutation in tumor biopsies with high sensitivity for clinical testing. Tumorplex™ is a novel modification of Sanger sequencing technology that generates both mutant and wild type nucleotide sequences simultaneously in the same electropherogram. The molecular weight of the two sequencing primers are different such that the two sequences generated are separated, thus eliminating possible suppression of mutant signal by the more abundant wild type signal. Tumorplex™ platform technology was tested using BRAF mutation V600E. These studies were performed with cloned BRAF mutations and genomic DNA extracted from tumor cells carrying 50% mutant allele. The lower limit of detection for BRAF V600E was found to be 20 genome equivalents (GE) using genomic DNA extracted from mutation specific cell lines. Sensitivity of the assay was tested by challenging the mutant allele with wild type allele at 20GE, and was able to detect BRAF mutant signal at a GE ration of 20:1×107 (mutant to wild-type). This level of sensitivity can detect low abundance of clonal mutations in tumor biopsies and eliminate the need for cell enrichment.•Tumorplex™ is a single tube assay that permits the recognition of mutant allele without suppression by wildtype signal.•Tumorplex™ provides a high level of sensitivity.•Tumorplex™ can be used with small sample size with mixed population of cells carrying heterogeneous gDNA.

Toehold-mediated strand displacement reaction triggered isothermal DNA amplification for highly sensitive and selective fluorescent detection of single-base mutation

Highly sensitive and selective detection strategy for single-base mutations is essential for risk assessment of malignancy and disease prognosis. In this work, a fluorescent detection method for single-base mutation was proposed based on high selectivity of toehold-mediated strand displacement reaction (TSDR) and powerful signal amplification capability of isothermal DNA amplification. A discrimination probe was specially designed with a stem-loop structure and an overhanging toehold domain. Hybridization between the toehold domain and the perfect matched target initiated the TSDR along with the unfolding of the discrimination probe. Subsequently, the target sequence acted as a primer to initiate the polymerization and nicking reactions, which released a great abundant of short sequences. Finally, the released strands were annealed with the reporter probe, launching another polymerization and nicking reaction to produce lots of G-quadruplex DNA, which could bind the N-methyl mesoporphyrin IX to yield an enhanced fluorescence response. However, when there was even a single base mismatch in the target DNA, the TSDR was suppressed and so subsequent isothermal DNA amplification and fluorescence response process could not occur. The proposed approach has been successfully implemented for the identification of the single-base mutant sequences in the human KRAS gene with a detection limit of 1.8pM. Furthermore, a recovery of 90% was obtained when detecting the target sequence in spiked HeLa cells lysate, demonstrating the feasibility of this detection strategy for single-base mutations in biological samples.

Gold nanostructures for the multiplex detection of glucose-6-phosphate dehydrogenase gene mutations

We describe a gold nanoparticle-based technique for the detection of single-base mutations in the glucose-6-phosphate dehydrogenase (G6PD) gene, a condition that can lead to neonatal jaundice and hemolytic anemia. The aim of this technique is to clearly distinguish different mutations frequently described within the Asian population from their wild-type counterparts and across different mutant variants. Gold nanoparticles of different sizes were synthesized, and each was conjugated with a single-strand DNA (ssDNA) sequence specific for a particular mutation in the G6PD gene. It was found that only mutant targets presented a characteristic band on the agarose gel, indicating the successful formation of dimeric nanostructures. No such dimer bands were observed for the wild-type targets. The difference in the relative dimer band levels allowed different mutant variants to be distinguished from one another. The technique was further validated using G6PD-deficient patient samples. This simple mutation detection method with direct result readout is amenable for rapid and mass screening of samples.

Quantitative detection of single base mutation by combining PNA hybridization and MALDI-TOF mass analysis

Peptide nucleic acid (PNA) probes were designed to bind to the internal reference sequence and the single base mutation sequence within PCR-amplified DNA templates. PNAs hybridized to the target sequences on DNA were analyzed using MALDI-TOF mass spectrometry. Accurate quantification of the relative amount of mutant DNA was reproducibly demonstrated.

Arabidopsis ENDO2: Its Catalytic Role and Requirement of N-Glycosylation for Function

The Arabidopsis thaliana At1g68290 gene encoding an endonuclease was isolated and designated ENDO2, which was cloned into a binary vector to overexpress ENDO2 with a C-terminal 6 × His-tag in A. thaliana. Our Arabidopsis transgenic lines harboring 35SP::ENDO2 produced stable active enzyme with high yield. The protein was affinity purified from transgenic plants, and its identity was confirmed by liquid chromatography-mass spectrometry and automatic Edman degradation. ENDO2 enzyme digests RNA, ssDNA, and dsDNA, with a substrate preference for ssDNA and RNA. The activity toward ssDNA (361.7 U/mg) is greater than its dsDNase activity (14.1 U/mg) at neutral pH. ENDO2 effectively cleaves mismatch regions in heteroduplex DNA containing single base pair mismatches or insertion/deletion bases and can be applied to high-throughput detection of single base mutation. Our data also validated that the removal of sugar groups from ENDO2 strongly affects its enzymatic stability and activity.