Fluorescent detection of single nucleotide polymorphism utilizing a hairpin DNA containing a nucleotide base analog pyrrolo-deoxycytidine as a fluorescent probe

A novel fluorescent method for the detection of single nucleotide polymorphism (SNP) was developed using a hairpin DNA containing nucleotide base analog pyrrolo-deoxycytidine (P-dC) as a fluorescent probe. This fluorescent probe was designed by incorporating a fluorescent P-dC into a stem of the hairpin DNA, whose sequence of the loop moiety complemented the target single strand DNA (ss-DNA). In the absence of the target ss-DNA, the fluorescent probe stays a closed configuration in which the P-dC is located in the double strand stem of the fluorescent probe, such that there is weak fluorescence, attributed to a more efficient stacking and collisional quenching of neighboring bases. In the presence of target ss-DNA, upon hybridizing the ss-DNA to the loop moiety, a stem-loop of the fluorescent probe is opened and the P-dC is located in the ss-DNA, thus resulting in strong fluorescence. The effective discrimination of the SNP, including single base mismatch ss-DNA (A, T, G) and double mismatch DNA (C, C), against perfect complementary ss-DNA was achieved by increased fluorescence intensity, and verified by thermal denaturation and circular dichroism spectroscopy. Relative fluorescence intensity had a linear relationship with the concentration of perfect complementary ss-DNA and ranged from 50 nM to 3.0 μM. The linear regression equation was F/F₀ = 2.73 C (μM) + 1.14 (R = 0.9961) and the detection limit of perfect complementary ss-DNA was 16 nM (S/N = 3). This study demonstrates that a hairpin DNA containing nucleotide base analog P-dC is a promising fluorescent probe for the effective discrimination of SNP and for highly sensitive detection of perfect complementary DNA.     

2-Amino-5,7-dimethyl-1,8-naphthyridine as a fluorescent reagent for the determination of nitrite

A new fluorescent reagent 2-amino-5,7-dimethyl-1,8-naphthyridine (ADMND) was proposed for the determination of trace nitrite. The reaction is based on the diazotization of naphthyridine amine with nitrite to form a diazonium salt that hydrolyzed when boiling to give hydroxyl group substituted naphthyridine. Fluorescence quenching degree of ADMND by nitrite ion is linear in the nitrite concentration range of 1 x 10(-7) to 2.5 x 10(-6)mol l(-1) with a detection limit of 4.06 x 10(-8)mol l(-1). Reaction and determination acidity for nitrite is the same which made the method much simpler compared with the widely accepted fluorescence method with DAN as a fluorescence reagent.     

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 novel fluorescent aptasensor based on silica nanoparticles,PicoGreen and exonuclease III as a signal amplification method for ultrasensitive detection of myoglobin

Measurement of myoglobin (Mb) in human blood serum is of great interest for quick diagnosis of acute myocardial infarction (AMI). In this study, a novel fluorescent aptasensor was designed for ultrasensitive and selective detection of Mb, based on target-induced high fluorescence intensity, complementary strand of aptamer (CS), PicoGreen (PG) dye, exonuclease III (Exo III) and silica nanoparticles coated with streptavidin (SNPs-Streptavidin). The developed aptasensor obtains characteristics of SNPs as enhancers of fluorescence intensity, Exo III as an enzyme which selectively digests the 3'-end of double-stranded DNA (dsDNA), PG as a fluorescent dye which could selectively bind to dsDNA and high selectivity and sensitivity of aptamer (Apt) toward its target. In the absence of Mb, no free CS remains in the environment of SNPs-Streptavidin, resulting in a weak fluorescence emission. In the present of Mb, dsDNA-modified SNPs-Streptavidin complex forms, leading to a very strong fluorescence emission. The developed fluorescent aptasensor exhibited high specificity toward Mb with a limit of detection (LOD) as low as 52 pM. In addition, the designed fluorescent aptasensor was efficiently used to detect Mb in human serum.     

Fluorescence-quenching phenomenon by photoinduced electron transfer between a fluorescent dye and a nucleotide base.

Fluorescently labeled oligonucleotide probes have been widely used in biotechnology, and fluorescence quenching by the interaction between the dyes and a nucleobase has been pointed out. This quenching causes big problem in analytical methods, but is useful in some other cases. Therefore, it is necessary to estimate the fluorescence quenching intensity under various conditions. We focused on the redox properties of some commercially available fluorescent dyes, and investigated dye-nucleotide interactions between a free dye and a nucleotide in aqueous solution by electrochemical and spectroscopic techniques. Our results suggested that the quenching was accompanied by photoinduced electron transfer between a thermodynamically quenchable excited dye and a specific base. Several kinds of fluorescent dyes labeled to the 5'-end of oligonucleotide C10T6 were prepared, and their quenching ratios compared upon hybridization with the complementary oligonucleotide A6G10. The quenching was completely reversible and their efficiencies depended on the attached fluorophore types. The fluorescence of 5-FAM, BODIPY FL or TAMRA-modified probe was strongly quenched by hybridization.     

Dual fluorophore PNA FIT-probes - extremely responsive and bright hybridization probes for the sensitive detection of DNA and RNA

Fluorescently labeled oligonucleotides are commonly employed as probes to detect specific DNA or RNA sequences in homogeneous solution. Useful probes should experience strong increases in fluorescent emission upon hybridization with the target. We developed dual labeled peptide nucleic acid probes, which signal the presence of complementary DNA or RNA by up to 450-fold enhancements of fluorescence intensity. This enabled the very sensitive detection of a DNA target (40 pM LOD), which was detectable at less than 0.1% of the beacon concentration. In contrast to existing DNA-based molecular beacons, this PNA-based method does not require a stem sequence to enforce dye-dye communication. Rather, the method relies on the energy transfer between a "smart" thiazole orange (TO) nucleotide, which requires formation of the probe-target complex in order to become fluorescent, and terminally appended acceptor dyes. To improve upon fluorescence responsiveness the energy pathways were dissected. Hydrophobic, spectrally mismatched dye combinations allowed significant (99.97%) decreases of background emission in the absence of a target. By contrast, spectral overlap between TO donor emission and acceptor excitation enabled extremely bright FRET signals. This and the large apparent Stokes shift (82 nm) suggests potential applications in the detection of specific RNA targets in biogenic matrices without the need of sample pre-processing prior to detection.     

Nucleic acid detection using carbon nanoparticles as a fluorescent sensing platform

In this communication, we demonstrate for the first time the proof of concept that carbon nanoparticles (CNPs) can be used as an effective fluorescent sensing platform for nucleic acid detection with selectivity down to single-base mismatch. The dye-labeled single-stranded DNA (ssDNA) probe is adsorbed onto the surface of the CNP via π-π interaction, quenching the dye. In the target assay, a double-stranded DNA (dsDNA) hybrid forms, recovering dye fluorescence.     

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.     

Twin probes as a novel tool for the detection of single-nucleotide polymorphisms

Single-nucleotide polymorphisms (SNPs) are the most common form of DNA sequence variation. There is a strong interest from both academy and industry to develop rapid, sensitive and cost effective methods for SNP detection. Here we report a novel structural concept for DNA detection based on fluorescence dequenching upon hybridization. The so-called "twin probe" consists of a central fluorene derivative as fluorophore to which two identical oligonucleotides are covalently attached. This probe architecture is applied in homogeneous hybridization assays with subsequent fluorescence spectroscopic analysis. The bioorganic hybrid structure is well suited for sequence specific DNA detection and even SNPs are identified with high efficiency. Additionally, the photophysical properties of the twin probe were investigated. The covalent attachment of two single stranded oligonucleotides leads to strong quenching of the central fluorescence dye induced by the nucleobases. The twin probe is characterized by supramolecular aggregate formation accompanied by red-shifted emission and broad fluorescence spectra.     

A pyrazine-based fluorescence-enhancing ligand with a high selectivity for thymine in AP site-containing DNA duplexes

A fluorescent pyrazine derivative, 3,5-diamino-6-chloro-2-pyrazine carbonitrile (DCPC), is presented as a promising light-up ligand for single-nucleotide polymorphisms (SNPs) typing. In solutions buffered to pH 7.0 (I = 0.11 M, at 5 degrees C), DCPC can bind to thymine selectively over other nucleobases opposite an abasic site in DNA duplexes (5'-GTGTG CGTTG ANA TGGAC GCAGA-3'/3'-CACAC GCAAC TXT ACCTG CGTCT-5', X = abasic site, N = target nucleotide) with a dissociation constant of 2.6 microM. The binding of DCPC is accompanied by a significant enhancement of its fluorescence (lambda(max), 412 nm), and the response is highly selective to thymine base. These binding and sensing properties allow a clear detection of thymine-related mutations present in polymerase chain reaction (PCR) amplification products.     

基于DNA双链取代策略免标记检测铅离子的研究

建立了一种基于DNA双链取代策略和SYBR GreenⅠ(SG)作为荧光染料插入剂进行免标记铅离子检测的荧光传感方法。SG作为一种染料分子,与单链DNA作用产生的荧光强度很弱,但可以插入双链DNA,使SG荧光强度明显增强。检测时铅离子适配体首先与其部分互补单链DNA杂交形成稳定的双链DNA结构,当溶液中存在铅离子时,铅离子与其适配体特异性结合,双链DNA的数量减少,加入SG可实现铅离子的免标记定量检测。此方法具有灵敏度高、特异性强、简便快速等优点。最低检测浓度为2 nmol/L,检出限(S/N=3)为1.6 nmol/L,实际样品检测结果良好。     

Affinity labeling of a single guanine bulge

We have developed a conceptually new method for the selective labeling of duplex DNA containing a guanine bulge with a masked form of fluorescent 2-amino-1,8-naphthyridine. A naphthyridine derivative 2 tethering DNA-alkylating epoxide was synthesized from (S)-epichlorohydrin and naphthyridine derivative 1, which selectively binds to the guanine bulge duplex. HPLC analysis of the labeling reaction of bulge duplex d(GTT GTGTTG GA)/d(CAA CA A ACC T) (TGT/A_A) with 2 showed a formation of 2-TGT adduct for the guanine bulge. The reaction proceeded for the guanine bulge and a reduced efficiency for the cytosine bulge, but not at all for adenine and thymine bulges. The site of covalent bond formation in 2-TGT was unambiguously identified at the guanine two bases away from the bulge by the use of MALDI-TOF MS analysis of the oligomer fragments produced by strand scission. The labeling reaction was also observed for the guanine bulge flanking two G-C base pairs (CGC/G_G), producing a 2:1 adduct (2.2-CGC). Upon hydrolysis of 2-TGT and 2.2-CGC with concentrated hydrogen chloride, a release of fluorescent 2-aminonaphthyridine from the adduct was clearly detected, verifying a concept of an affinity labeling of the guanine bulge with a masked fluorescent chromophore. The affinity labeling targeting of the guanine bulge is a conceptually novel method for the postsynthetic labeling of DNA. Hybridization, to the target sequence, of a probe DNA possessing one extra guanine especially between two cytosines provides a unique site for the labeling by masked fluorophore 2. The technique may have broad application in genetic typing without using a conventional synthesis of fluorescent-labeled DNA oligomers.     

Genotyping of two single nucleotide polymorphisms in 5,10-methylenetetrahydrofolate reductase by multiplex polymerase chain reaction and capillary electrophoresis

Two single nucleotide polymorphisms (SNPs) of 5,10-methylenetetrahydrofolate reductase (MTHFR) gene, A1298C and C677T, were widely considered to be related with various neoplasia disorders. We established a simple and effective capillary electrophoresis (CE) method for detection of two SNPs in MTHFR gene simultaneously. DNA samples were amplified by multiplex PCR with universal fluorescence-labeled primer and analyzed by single-strand conformation polymorphism (SSCP)-CE method. The CE method was performed using 1.5% hydroxyethyl cellulose in 1× TBE buffer containing 1 M urea. The PCR products after SSCP procedure were electrokinetically injected at −10 kV, 30 s. Separation voltage was −6 kV and the temperature was set at 20 °C. The optimal SSCP-CE method was applied to detect two polymorphisms in MTHFR gene of acute lymphoblastic leukemia (ALL) and attention-deficit/hyperactivity disorder (ADHD) patients. Genotyping results were evaluated in terms of relationships between outcomes for ADHD patients after ALL chemotherapy and ALL disease. The SSCP-CE method and multiplex PCR with universal fluorescence primer were used as the fast technique for screening two SNPs in MTHFR gene, A1298C and C677T. The genotyping data were coincident with DNA sequencing. This SSCP-CE method was found feasible for detecting mutation of MTHFR gene in populations.     

PNA for one-base differentiating protection of DNA from nuclease and its use for SNPs detection

By the combination of peptide nucleic acid (PNA) with single-stranded DNA specific nucleases, alteration of a single base to another in DNA has been detected with high accuracy. Only the DNAs in DNA/PNA duplexes involving a mismatch are efficiently hydrolyzed by these enzymes, whereas fully matching sequences are kept intact. This difference is visually scored by adding 3,3'-diethylthiadicarbocyanine, which changes its color from blue to purple upon binding to DNA/PNA duplexes. These findings are applied to the convenient and straightforward detection of single nucleotide polymorphisms (SNPs). When the target site in the sample DNA is completely complementary with the PNA, a notable amount of DNA/PNA duplex remains and thus the solution exhibits purple color. In the presence of even one mismatch between PNA and DNA, however, the DNA is completely digested by the enzyme and therefore the dye shows its intrinsic blue color. The SNPs in the apolipoprotein E gene of human DNA have been successfully genotyped by this method.     

ECHO probes: a concept of fluorescence control for practical nucleic acid sensing

An excitonic interaction caused by the H-aggregation of fluorescent dyes is a new type of useful photophysical process for fluorescence-controlled nucleic acid sensing. This critical review points out the recent advances in exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probes, which have a fluorescence-labeled nucleotide in which two molecules of thiazole orange or its derivatives are linked covalently. ECHO probes show absorption shift and emission switching depending on hybridization with the target nucleic acid. The hybridization-sensitive fluorescence emission of ECHO probes and the further modification of probes have made possible a variety of practical applications, such as multicolor RNA imaging in living cells and facile detection of gene polymorphism (144 references).     

Ag nanoclusters as probes for turn-on fluorescence recognition of TpG dinucleotide with a high selectivity

CpG dinucleotide in DNA has a great tendency to mutate to TpG dinucleotide and this transition can cause some serious diseases. In this work, fluorescent Ag nanoclusters (Ag NCs) were employed as useful inorganic fluorophores for the potential of selectively discriminating TpG dinucleotide from CpG dinucleotide. Opposite the base Y of interest in YpG dinucleotide (Y = C or T), a bulge site was introduced so as to make the base Y to be unpaired and ready for Ag⁺ binding. Such that the unpaired Y and context base pairs can provide a specific space suitable for creating fluorescent Ag NCs. We found that in comparison with CpG dinucleotide, TpG dinucleotide is much more efficient in growing fluorescent Ag NCs. Therefore, mutation of CpG dinucleotide to TpG can be identified by a turn-on fluorescence response and a high selectivity. More interestingly, Ag NCs exhibit a better performance in the TpG recognition over the other dinucleotides (Y = A and G) than the previously used organic fluorophores. Additionally, the effectiveness of the bulge site design in discriminating these dinucleotides was evidenced by control DNAs having the abasic site structure. We expect that a practical method for TpG dinucleotide recognition with a high selectivity can be developed using the bulge site-grown fluorescent Ag NCs as novel probes.     

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.     

Detection of single-molecule DNA hybridization by using dual-color total internal reflection fluorescence microscopy

We examined the use of prism-type simultaneous dual-color total internal reflection fluorescence microscopy (TIRFM) to probe DNA molecules at the single-molecule level. The system allowed the direct detection of the complementary interactions between single-stranded probe DNA molecules (16-mer) and various lengths of single-stranded target DNA molecules (16-mer and 55-mer) that had been labeled with different fluorescent dyes (Cy3, Cy5, and fluorescein). The polymer-modified glass substrate and the extent of DNA probe immobilization were easily characterized either with standard TIRFM or with atomic force microscopy. However, only dual-color TIRFM could provide unambiguous images of individual single-stranded target DNA molecules hybridized with the correct sequence in the range of fM–aM. Succinic anhydride showed low RMS roughness and was found to be an optimal blocking reagent against non-specific adsorption, with an efficiency of 92%. This study provides a benchmark for directly monitoring the interactions and the detection of co-localization of two different DNA molecules and can be applied to the development of a nanoarray biochip at the single-molecule level.     

Rapid DNA hybridization analysis using a PDMS microfluidic sensor and a molecular beacon.

A rapid DNA analysis has been developed based on a fluorescence intensity change of a molecular beacon in a PDMS microfluidic channel. Recently, we reported a new analytical method of DNA hybridization involving a PDMS microfluidic sensor using fluorescence energy transfer (FRET). However, there are some limitations in its application to real DNA samples because the target DNA must be labelled with a suitable fluorescent dye. To resolve this problem, we have developed a new DNA microfluidic sensor using a molecular beacon. By monitoring the change in the restored fluorescence intensity along the channel length, it is possible to rapidly detect any hybridization of the molecular beacon to the target DNA. In this case, the target DNA does not need to be labelled. Our experimental results demonstrate that this microfluidic sensor using a molecular beacon is a promising diagnostic tool for rapid DNA hybridization analysis.