Detection Of Single-nucleotide Mismatches Research Articles

Activated single nucleotide mismatch determination through toehold-embedded hairpin-mediated strand displacement reaction alongside CRISPR-Cas12a for detection of TP53 point mutation

The CRISPR-Cas12a-based diagnostic platform is renowned for its high sensitivity and, when coupled with the toehold-mediated strand displacement (TMSD) reaction, enables the detection of single nucleotide mismatch (SNM). However, conventional TMSD with the CRISPR-Cas12a system suffers from signal leakage due to overlapping activator sequences and low displacement efficiency. Here, we propose a toehold-embedded hairpin-mediated strand displacement (THMSD) method and leverage the magnesium attenuation effect of CRISPR-Cas12a for selective single-point mutation detection in the TP53 gene. This approach effectively mitigates signal leakage as the hairpin DNA retains partial crRNA targeting sequences within its loop, preventing sequence overlap with the trigger and direct Cas12a activation. Additionally, the magnesium attenuation effect reduces background signal and enhances selectivity. We investigated toehold length and mismatch position to detect SNM with a high discrimination factor (DF). This high DF allows the THMSD/CRISPR-Cas12a system to differentiate between TP53 R273H mutant and TP53 wildtype genes for cancer screening at low abundances, down to 0.1 % with a concentration of 20 pM. When used for screening between H1975 and WI38 cells, the statistically significant difference was attainable with polymerase chain reaction (PCR) template loading as low as 4 ng. These results pave the way for developing CRISPR-Cas12a-based methods to effectively detect TP53 mutations as lung cancer biomarkers.

A kinetically controlled, isothermal method for the detection of single nucleotide mismatches

We describe an isothermal, enzyme-free method to detect single nucleotide differences between oligonucleotides of close homology. The approach exploits kinetic differences in toe-hold-mediated, nucleic acid strand-displacement reactions to detect single nucleotide polymorphisms (SNPs) with essentially “digital” precision. The theoretical underpinning, experimental analyses, predictability, and accuracy of this new method are reported. We demonstrate detection of biologically relevant SNPs and single nucleotide differences in the let-7 family of microRNAs. The method is adaptable to microarray formats, as demonstrated with on-chip detection of SNP variants involved in susceptibility to the therapeutic agents abacavir, Herceptin, and simvastatin.

Highly specific SNP detection using 2D graphene electronics and DNA strand displacement

Single-nucleotide polymorphisms (SNPs) in a gene sequence are markers for a variety of human diseases. Detection of SNPs with high specificity and sensitivity is essential for effective practical implementation of personalized medicine. Current DNA sequencing, including SNP detection, primarily uses enzyme-based methods or fluorophore-labeled assays that are time-consuming, need laboratory-scale settings, and are expensive. Previously reported electrical charge-based SNP detectors have insufficient specificity and accuracy, limiting their effectiveness. Here, we demonstrate the use of a DNA strand displacement-based probe on a graphene field effect transistor (FET) for high-specificity, single-nucleotide mismatch detection. The single mismatch was detected by measuring strand displacement-induced resistance (and hence current) change and Dirac point shift in a graphene FET. SNP detection in large double-helix DNA strands (e.g., 47 nt) minimize false-positive results. Our electrical sensor-based SNP detection technology, without labeling and without apparent cross-hybridization artifacts, would allow fast, sensitive, and portable SNP detection with single-nucleotide resolution. The technology will have a wide range of applications in digital and implantable biosensors and high-throughput DNA genotyping, with transformative implications for personalized medicine.

Electrochemical signature of mismatch in overhang DNA films: a scanning electrochemical microscopic study

High throughput DNA basepair mismatch detection is an ultimate goal for earlier and point-of-care diagnostics. However, the size of a target sequence on single nucleotide mismatch detection will critically impact the design of sensors in future. To study the potential impact of target size, the probe and target strands of unequal size were hybridized in the absence and presence of single nucleotide mismatches along the sequence. After hybridization, the shorter target sequences form overhangs in the probe strand while longer target sequences form overhangs in the complementary strand. The resulting double stranded DNA hybrids were printed on gold surfaces and the electrochemical response of the films was studied by scanning electrochemical microscopy without signal amplification and label. The redox mediator, [Fe(CN)(6)](4-), experiences lower repulsion in the vicinity of mismatch containing ds-DNA films, which ultimately manifests into higher feedback current regardless of the size and hybridization position of the complementary strands. Kinetic rate constants monitored right above the ds-DNA films show k(0) = 4.5 ± 0.1 × 10(-5) cm s(-1) for the short sequence hybridized at the upper portion of the probe while k(0) = 4.1 ± 0.2 × 10(-5) cm s(-1) for longer complementary strands which has only top overhang. It suggests that hybridization position is important for mismatch detection in short complementary stands. However, in longer complementary strands, mismatches are easily detectable in the absence of bottom overhangs.

Scanning positional variations in single-nucleotide polymorphism of DNA: an electrochemical study

While there are a number of electrochemical methods reported that enable the detection of single nucleotide mismatches, the determination of mismatch position in a double stranded DNA remains an unsolved challenge. Using a model system, we systematically explored the electrochemical response of all possible positions of single nucleotide mismatches in a set of 25-mer DNA films. These ds-DNA sequences each with a single mismatch at one of the twenty-five positions were bound to gold surfaces through a Au-S linkage and analyzed by electrochemical impedance spectroscopy (EIS) and scanning electrochemical microscopy (SECM) in the absence and presence of Zn(2+). We expected a unique response from each mismatched sequence in order to discriminate the mismatch positions. A pattern emerges between the electrochemical signals and mismatch positions. The positions can be grouped broadly into positions that exhibit large differences between matched and mismatched DNA (around positions 5 and 9) and those that exhibit smaller differences (around positions 1, 13 and 23) in the charge transfer resistance ΔR(ct), evaluated by EIS, and the apparent rate constant k(0), evaluated by SECM. To the best of our knowledge, this is the first study evaluating the electrochemical response of a single nucleotide mismatch as a function of mismatch positions along an oligonucleotide sequence.

Large flanking sequence effects in single nucleotide mismatch detection using fluorescent nucleoside Çf

The first systematic study of flanking sequence effects on mismatch detection by a fluorescent nucleotide is described, using fluoroside C(f). Although a high degree of variance was observed in fluorescence intensity of mismatched duplexes between different flanking sequences, C(f) was able to distinguish a mismatch from the fully base-paired duplex in 13 out of 16 sequences, and even identify each mismatch in 10 of those flanking sequences. For the flanking sequences where fluoroside C(f) did not unambiguously determine its base-pairing partner, the experimental conditions were varied in an attempt to facilitate mismatch identification. No beneficial effect on the relative fluorescence intensities was achieved by changing the temperature, adding organic co-solvents or potassium iodide. In contrast, mercuric ions selectively quenched the fluorescence intensity of the C(f).T mismatch, effectively resolving the overlap of all emission spectra and thereby facilitating identification of all base-pairing partners in any flanking sequence by C(f). This is the first time mercuric ions have been used to selectively quench the fluorescence of a single mismatch. A noticeable characteristic of C(f) is that, unlike most fluorosides, the fluorescence intensity of C(f) was not quenched to a discernable degree by a flanking G-C pair.

Detection of oligonucleotide systematic mismatches with a surface plasmon resonance sensor

The detection and parallel characterization of the hybridization event of 21-base, unlabeled DNA oligonucleotides with a monolayer of complementary DNA immobilized on a gold surface by surface plasmon resonance (SPR) is presented. A thiol modification on the probe DNA strand allowed for its attachment to the surface via self-assembly. For the hybridization of full match DNA a detection limit of 20 pM was determined. The change in SPR signal was always larger for the full match compared to the one-mismatch target DNA oligonucleotides when the mismatch was in the middle or at the proximal end of the target DNA. Hybridization conditions (buffer concentration, flow rate, and temperature) did not affect the ability of the sensor to discriminate for single nucleotide mismatches. To our knowledge this is the only work where a comparison on the surface hybridization efficiency is performed between proximal, distal, and middle mismatches and the effect of three hybridization parameters is studied with regard to the detection of single nucleotide mismatches using SPR.

Label-Free DNA Sequence Detection with Enhanced Sensitivity and Selectivity Using Cationic Conjugated Polymers and PicoGreen

In this Letter, we demonstrate that PicoGreen can be used in combination with cationic conjugated polymers to develop label-free DNA sensors with enhanced detection efficiency and further improved selectivity. The high selectivity enables detection of single nucleotide mismatch even at room temperature. This method uses all commercially available materials. It is low cost, is simple to use, and works in a "mix-and-detect" manner. The detection sensitivity could be improved by a factor of 19 times through resonance energy transfer using poly[(9,9-di(3,3'-N,N'-trimethyl-ammonium)propylfluorenyl-2,7-diyl)-alt-co-(1,4-phenylene)] diiodide salt (PFP) as a light harvesting complex, to take advantage of its collective optical response and optical amplification effects. The further improved selectivity is ascribed to the stronger binding interaction between PicoGreen with dsDNA compared to that between PicoGreen and ssDNA, which results in selective repelling of PicoGreen away from the DNA strands.

2′‐N‐(Pyren‐1‐yl)acetyl‐2′‐Amino‐α‐L‐LNA: Synthesis and Detection of Single Nucleotide Mismatches in DNA and RNA Targets

2′‐<i>N</i>‐(Pyren‐1‐yl)acetyl‐2′‐Amino‐α‐L‐LNA: Synthesis and Detection of Single Nucleotide Mismatches in DNA and RNA Targets

Protons and single nucleotide polymorphism detection: A simple use for the Ion Sensitive Field Effect Transistor

The identification of single nucleotide polymorphisms (SNP) is a subject receiving considerable attention owing to the possibility of diagnosing and screening for diseases they are connected with. Currently, the emphasis for their detection is using DNA hybridisation methods and the development of novel, mainly fluorescent based methods for hybridisation detection. We report on a new method for identifying SNPs using Ion Sensitive Field Effect Transistor technology. Through the detection of single nucleotide mismatches, we show a procedure for effectively sequencing one base and therefore a mechanism for sequencing new DNA.

UV light-aided immobilization of oligonucleotides on glass surface using N -(3-trifluoroethanesulfonyloxypropyl)anthraquinone-2-carboxamide (NTPAC) and detection of single nucleotide mismatches

UV light-aided immobilization of oligonucleotides on glass surface using N -(3-trifluoroethanesulfonyloxypropyl)anthraquinone-2-carboxamide (NTPAC) and detection of single nucleotide mismatches