High-throughput single nucleotide polymorphism typing by fluorescent single-strand conformation polymorphism analysis with capillary electrophoresis

In this study, we performed high-throughput and precise single nucleotide polymorphism (SNP) typing by fluorescent capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) analysis. A system composed of a multicapillary DNA analyzer, a newly developed sieving matrix, four different colors of fluorescent labels, and a multiplex polymerase chain reaction (PCR) enabled low-cost and highly reliable SNP typing. Moreover, this system enabled the estimation of SNP allele frequencies using pooled DNA samples, which should be beneficial for large-scale association studies. Thus, fluorescent CE-SSCP analysis is a useful method for large-scale SNP typing.     

Novel fluorescent colloids as a DNA fluorescence probe

Fluorescent perylene colloids in the 80–90 nm size range have been prepared by the reprecipitation method. These nanoparticles were modified by cetyltrimethylammonium bromide (CTAB) which inhibited their growth. The nanoparticles also readily interacted with DNA. The fluorescence emission was measured at _ex/_em=400/565 nm. The fluorescence decrease of colloid–CTAB in aqueous solution was measured in the presence of nucleic acids. Under the optimum conditions, the ratio of fluorescence intensity in the absence and presence of nucleic acids was proportional to the concentration of nucleic acids over the range 0.02–5.1 µg mL^–1 for FS (fish sperm) DNA or CT (calf thymus) DNA. The detection limits were 0.01 µg mL^–1 for FS DNA and 0.012 µg mL^–1 for CT DNA, respectively. Based on this approach, a new quantitative method for DNA assay is presented in this paper.     

Non‐hybridization single nucleotide polymorphism detection and genotyping assay through direct discrimination of single base mutation by capillary electrophoretic separation of single‐stranded DNA

The significant demands for single nucleotide polymorphism detection and genotyping assays have grown. Most common assays are based on the recognition of the target sequence by the hybridization with its specific probe having the complementary sequence of the target. Herein, a simple, label‐free, and economical non‐hybridization assay was developed for single nucleotide polymorphism detection and genotyping, based on the direct discrimination of single base mutation by simple capillary electrophoresis separation for single‐stranded DNA in an acidic electrophoretic buffer solution containing urea. Capillary electrophoresis separation of single‐base sequential isomers of DNA was achieved due to charge differences resulting from the different protonation properties of the DNA bases. Single nucleotide polymorphism detection and genotyping were achieved by discriminating the electropherogram pattern change, that is, peak number in the electropherogram, obtained by the proposed method. The successful practical application of the proposed method was demonstrated through single nucleotide polymorphism detection and genotyping on a known gene region of 84‐mer, in which guanine to adenine single‐base mutation is commonly observed, using a human hair sample in combination with genomic DNA extraction, polymerase chain reaction amplification, DNA purification from polymerase chain reaction products, and capillary electrophoresis separation.     

Development of a light-up fluorescent probe for HRAS G-quadruplex DNA

The development of small-molecule G-quadruplex DNA probes has attracted significant attention in recent years. However, G-quadruplexes can display a wide variety of topologies, which process different structures and functions. Therefore, selective discrimination one G-quadruplex structure over another is promising. Herein, we reported the design, synthesis and biological evaluation of a long-chain fatty amine functionalized triphenylamine-quinolinium conjugate 1b. Significant enhancement of the fluorescence intensity (over 180 fold) was observed when 1b bound with HRAS G-quadruplex DNA, while much weaker enhancements were presented in the presence of other G-quadruplexes (45–90-fold) and single/double-stranded DNAs (less than 20-fold), indicating 1b had an excellent selectivity to HRAS. The details of the interactions were investigated by UV–Vis, FID and CD analysis. The results show 1b could interact and stabilize HRAS structure mainly by π-π stacking binding mode. The introduced amine chain of the structure core was found to be better in the terms of inducing selectivity toward G-quadruplex structure. In addition, the application of 1b as a fluorescent agent for living cell imaging was also demonstrated.     

Highly sensitive fluorescent probe for detection of alkaloids

A new fluorescent probe, based on an amphiphilic Schiff-base zinc(II) complex, 1, for the sensitive detection of some important classes of alkaloids is presented. It exhibits optical absorption changes and fluorescence enhancement upon formation of 1:1 1·alkaloid adducts. Four diverse classes of alkaloids, represented by their basic structures and related representative prototypes, are investigated, through the study of optical and binding properties of 1·alkaloid adducts. It is found that the chromogenic and fluorogenic complex 1 is selective between these classes of alkaloids in the micromolar range, with a limit of quantification of 0.40 μM for nicotine and 0.43 μM for cinchonine.     

Estimation of the optimal electrophoretic temperature of DNA single-strand conformation polymorphism by DNA base composition

To explore the relation between DNA base composition and the optimal single-strand conformation polymorphism (SSCP) electrophoretic temperature (T(s)), we analyzed DNA base composition and T(s) of 24 DNA fragments from different genes and found that T(s) was positively correlative with the ratio of base C/base A. T(s) could be estimated by the formula T(s) = [80 x C/(A+1)]/[2.71 + [C/(A+1)]]. T(s) could be increased dramatically by the complementary sequences in both 5'-and 3'-ends of a DNA single-strand.     

A self-assembled deoxyribonucleic acid concatemer for sensitive detection of single nucleotide polymorphism

Polymerase-free and label-free strategies for DNA detection have shown excellent sensitivity and specificity in various biological samples. Herein, we propose a method for single nucleotide polymorphism (SNP) detection by using self-assembled DNA concatemers. Capture probes, bound to magnetic beads, can joint mediator probes by T4 DNA ligase in the presence of target DNA that is complementary to the capture probe and mediator probe. The mediator probes trigger self-assembly of two auxiliary probes on magnetic beads to form DNA concatemers. Separated by a magnetic rack, the double-stranded concatemers on beads can recruit a great amount of SYBR Green I and eventually result in amplified fluorescent signals. In comparison with reported methods for SNP detection, the concatemer-based approach has significant advantages of low background, simplicity, and ultrasensitivity, making it as a convenient platform for clinical applications. As a proof of concept, BRAF^T1799A oncogene mutation, a SNP involved in diverse human cancers, was used as a model target. The developed approach using a fluorescent intercalator can detect as low as 0.1 fM target BRAF^T1799A DNA, which is better than those previously published methods for SNP detection. This method is robust and can be used directly to measure the BRAF^T1799A DNA in complex human serum with excellent recovery (94–103%). It is expected that this assay principle can be directed toward other SNP genes by simply changing the mediator probe and auxiliary probes.     

2-Aminopurine hairpin probes for the detection of ultraviolet-induced DNA damage

Nucleic acid exposure to radiation and chemical insults leads to damage and disease. Thus, detection and understanding DNA damage is important for elucidating molecular mechanisms of disease. However, current methods of DNA damage detection are either time-consuming, destroy the sample, or are too specific to be used for generic detection of damage. In this paper, we develop a fluorescence sensor of 2-aminopurine (2AP), a fluorescent analogue of adenine, incorporated in the loop of a hairpin probe for the quantification of ultraviolet (UV) C-induced nucleic acid damage. Our results show that the selectivity of the 2AP hairpin probe to UV-induced nucleic acid damage is comparable to molecular beacon (MB) probes of DNA damage. The calibration curve for the 2AP hairpin probe shows good linearity (R² = 0.98) with a limit of detection of 17.2 nM. This probe is a simple, fast and economic fluorescence sensor for the quantification of UV-induced damage in DNA.     

Detection of DNA base variation and cytosine methylation at a single nucleotide site using a highly sensitive fluorescent probe

A fluorescent DNA probe containing an anthracene group attached via an anucleosidic linker can identify all four DNA bases at a single site as well as the epigenetic modification C/5-MeC via a hybridisation sensing assay.     

Effect of oligonucleotide probes substituted by deoxyinosines on the specificity of SNP detection on the DNA microarray

One of the main factors that can affect the quality of microarray results is the microarray hybridization specificity. The key factor that affects hybridization specificity is the design of the probes. In this paper, we described a novel oligonucleotide probe containing deoxyinosines aimed at improving DNA hybridization specificity. We compared different probes to determine the distance between deoxyinosine base and SNPs site and the number of deoxyinosine bases. The new probe sequences contained two set of deoxyinosines (each set had two deoxyinosines), in which the interval between SNP site and each set of deoxyinosines was two bases. The new probes could obtain the highest hybridization specificity. The experimental results showed that probes containing deoxyinosines hybridized effectively to the perfectly matched target and improved the hybridization specificity of DNA microarray. By including a simple washing step after hybridization, these probes could distinguish matched targets from single‐base‐mismatched sequences perfectly. For the probes containing deoxyinosines, the fluorescence intensity of a match sequence was more than eight times stronger than that of a mismatch. However, the intensity ratio was only 1.3 times or less for the probes without deoxyinosines. Finally, using hybridization of the PCR product microarrays, we successfully genotyped SNP of 140 samples using these new labeled probes. Our results show that this is a useful new strategy for modifying oligonucleotide probes for use in DNA microarray analysis.     

A coumarin-based fluorescent probe for selective detection of Cu2+ in water

Fluorescent Red GK, a commercially available coumarin-based dye, was developed as a “turn-off” fluorescent probe for detection of Cu²⁺ in aqueous solution. It exhibited high selectivity and sensitivity at room temperature. Upon addition of Cu²⁺, the strong fluorescence of Fluorescent Red GK was severely quenched and its color changed from orange to colorless under illumination with a UV lamp; the color of the solution also changed from pink to colorless. So, it can be used as a specific colorimetric and fluorescent probe for Cu²⁺ with a detection limit as low as 0.0634?μM.     

Evaluation of a potential epigenetic biomarker by quantitative methyl-single nucleotide polymorphism analysis

Tumorigenesis is characterized by alterations of methylation profiles including loss and gain of 5-methylcytosine. Recently, we identified a single CpG, which seemed to be consistently hypomethylated in pilocytic astrocytomas but not in other gliomas. To evaluate its applicability as a biomarker, we examined its methylation status in a large panel of gliomas (n = 97). Methylation-dependent DNA sequence variation may be considered a kind of single nucleotide polymorphism (methylSNP). MethylSNPs can be easily converted into common SNPs of the C/T type by sodium bisulfite treatment of the DNA and afterwards subjected to conventional SNP typing. We adapted SnaPshot trade mark and Pyrosequencing trade mark to determine the methylation of our test CpG in a quantitative manner. The adapted methods, called SNaPmeth and PyroMeth, respectively, gave nearly identical results, however data obtained with PyroMeth showed less scattering. Furthermore, the integrated software for allele frequency determination from Pyrosequencing could be used directly for data analysis while SnaPmeth data had to be exported and processed manually. Although data did not confirm our previous result of a preferential hypomethylation of the tested CpG in pilocytic astrocytomas, we consider quantitative methylSNP analysis by SNaPmeth or PyroMeth a favorable alternative to existing high-throughput methylation assays. It combines single CpG analysis with accurate quantitation and is amenable to high throughput.     

Magnetic porous carbon nanocomposites derived from metal-organic frameworks as a sensing platform for DNA fluorescent detection

Metal-organic frameworks (MOFs) have emerged as very fascinating functional materials due to their tunable nature and diverse applications. In this work, we prepared a magnetic porous carbon (MPC) nanocomposite by employing iron-containing MOFs (MIL-88A) as precursors through a one-pot thermolysis method. It was found that the MPC can absorb selectively single-stranded DNA (ssDNA) probe to form MPC/ssDNA complex and subsequently quench the labelled fluorescent dye of the ssDNA probe, which is resulted from the synergetic effect of magnetic nanoparticles and carbon matrix. Upon the addition of complementary target DNA, however, the absorbed ssDNA probe could be released from MPC surface by forming double-stranded DNA with target DNA, and accompanied by the recovery of the fluorescence of ssDNA probe. Based on these findings, a sensing platform with low background signal for DNA fluorescent detection was developed. The proposed sensing platform exhibits high sensitivity with detection limit of 1 nM and excellent selectivity to specific target DNA, even single-base mismatched nucleotide can be distinguished. We envision that the presented study would provide a new perspective on the potential applications of MOF-derived nanocomposites in biomedical fields.     

A sensitive fluorescence anisotropy method for point mutation detection by using core-shell fluorescent nanoparticles and high-fidelity DNA ligase

The present study reports a proof-of-principle for a sensitive genotyping assay approach that can detect single nucleotide polymorphisms (SNPs) based on fluorescence anisotropy measurements through a core-shell fluorescent nanoparticles assembly and ligase reaction. By incorporating the core-shell fluorescent nanoparticles into fluorescence anisotropy measurements, this assay provided a convenient and sensitive detection assay that enabled straightforward single-base discrimination without the need of complicated operational steps. The assay was implemented via two steps: first, the hybridization reaction that allowed two nanoparticle-tagged probes to hybridize with the target DNA strand and the ligase reaction that generated the ligation between perfectly matched probes while no ligation occurred between mismatched ones were implemented synchronously in the same solution. Then, a thermal treatment at a relatively high temperature discriminated the ligation of probes. When the reaction mixture was heated to denature the duplex formed, the fluorescence anisotropy value of the perfect-match solution does not revert to the initial value, while that of the mismatch again comes back as the assembled fluorescent nanoparticles dispart. The present approach has been demonstrated with the discrimination of a single base mutation in codon 12 of a K-ras oncogene that is of significant value for colorectal cancers diagnosis, and the wild type and mutant type were successfully scored. Due to its ease of operation and high sensitivity, it was expected that the proposed detection approach might hold great promise in practical clinical diagnosis.     

A New Trace Analytic Method for the Determination of Sequence-Specific DNA with Fluorescent Probes

A new method of fluorescence spectrometry detection of single-strand DNA (ssDNA) was established by hybridizing the ssDNA with its complementary ssDNA to form double-stranded DNA (dsDNA). Our results show that the fluorescence intensity increased significantly when the nucleic acid molecular “light switch"(Ru(phen)₂dppx²⁺) or Hoechst 33258 dye interacted with dsDNA, and the fluorescence intensity also increased as the DNA concentration increased. The changing law was also studied about how the fluorescence intensity changed when the two kinds of fluorescent probes interacted with oligonucleotide of different lengths and different sequences, as well as DNA-DNA′ hybridization products. Then, the effect of the bases mismatch, varying length of DNA chain, and different DNA sequences on the fluorescence intensity were explored at the same time, by detecting the specific DNA sequence of avian influenza H1N1 virus, cauliflower mosaic virus, and hepatitis C virus. Additionally, the selectivity, linear range, and sensitivity of the two probes were compared.     

Quantum dot-based "turn-on" fluorescent probe for detection of zinc and cadmium ions in aqueous media

Health or environmental issue caused by abnormal level of metal ions like Zn²⁺ or Cd²⁺ is a worldwide concern. Developing an inexpensive and facile detection method for Zn²⁺ and Cd²⁺ is in urgent demand. Due to their super optical properties, fluorescent quantum dots (QDs) have been developed as a promising alternative for organic dyes in fluorescence analysis. In this study, a CdTe QDs-based sensitive and selective probe for Zn²⁺ and Cd²⁺ in aqueous media was reported. The proposed probe worked in fluorescence “turn-on” mode. The initial bright fluorescence of CdTe QDs was effectively quenched by sulfur anions (S²⁻). The presence of Zn²⁺ (or Cd²⁺) can “turn-on” the weak fluorescence of QDs quenched by S²⁻ due to the formation of ZnS (or CdS) passivation shell. Under optimal conditions, a good linear relationship between the fluorescence response and concentration of Zn²⁺ (or Cd²⁺) could be obtained in the range from 1.6 to 35 μM (1.3–25 μM for Cd²⁺). The limit of detection (LOD) for Zn²⁺ and Cd²⁺ were found to be 1.2 and 0.5 μM, respectively. Furthermore, the present probe exhibited a high selectivity for Zn²⁺ and Cd²⁺ over other metal ions and was successfully used in the detection of Zn²⁺ or Cd²⁺ in real water samples.     

Site-specific binding of chelerythrine and sanguinarine to single pyrimidine bulges in hairpin DNA

Spectrofluorometric titration, electrospray ionization time-of-flight mass spectrometric and UV melting methods were employed to study the binding of chelerythrine and sanguinarine to bulged DNA. The results showed that both alkaloids bind specifically to single pyrimidine (C, T) bulge sites. The ability of sanguinarine to bind to both regular and bulged hairpins was found to be stronger than that of chelerythrine, but the binding selectivity of chelerythrine toward single-base bulges was much larger than that of sanguinarine. Figure Association constants for chelerythrine and sanguinarine toward regular and single-base bulged hairpins obtained from fluorometric analysis     

Coupling hybridization chain reaction with DNAzyme recycling for enzyme-free and dual amplified sensitive fluorescent detection of methyltransferase activity

Aberrant DNA methylation originated from changes in DNA methyltransferase activity can lead to many genetic diseases and tumor types, and the monitoring of methyltransferase activity is thus of great importance in disease diagnosis and drug screening. In this work, by combing hybridization chain reaction (HCR) and metal ion-dependent DNAzyme recycling, we have developed a convenient enzyme-free signal amplification strategy for highly sensitive detection of DNA adenine methyltransferase (Dam MTase) activity and its inhibitors. The Dam MTase-induced methylation and subsequent cleavage of the methylated hairpin DNA probes by DpnI endonuclease lead to the release of ssDNA triggers for HCR formation of many Mg²⁺-dependent DNAzymes, in which the fluorescently quenched substrate sequences are catalytically and cyclically cleaved by Mg²⁺ to generate remarkably amplified fluorescent signals for highly sensitive detection of Dam MTase at 7.23 × 10⁻⁴ U/mL. In addition, the inhibition of different drugs to Dam MTase activity can also be evaluated with the developed method. With the advantages of simplicity and significant signal amplification over other common methods, the demonstrated biosensing approach thus offers great potential for highly sensitive detection of various methyltransferases and provides a convenient platform for drug screening for therapeutic applications.