Visual detection of Hg²⁺ in aqueous solution using gold nanoparticles and thymine-rich hairpin DNA probes

We report a sensitive method for visual detection of mercury ions (II) (Hg2?) in aqueous solution by using gold nanoparticles (Au-NPs) and thymine (T)-rich hairpin DNA probes. The thiolated hairpin DNA probe was immobilized on the Au-NP surface through a self-assembling method. Another thymine-rich, digoxin-labeled DNA probe was introduced to form DNA duplexes on the Au-NP surface with thymine-Hg2?-thymine (T-Hg2?-T) coordination in the presence of Hg2?. The Au-NPs associated with the formed duplexes were captured on the test zone of a lateral flow strip biocomponent (LFSB) by immunoreaction events between the digoxin on the duplexes and anti-digoxin antibodies on the LFSB. The accumulation of Au-NPs produced a characteristic red band on the test zone, enabling visual detection of Hg2? without instrumentation. A detection limit of 0.1 nM was obtained under optimal experimental conditions. This method provides a simple, rapid, sensitive approach for the detection of Hg2? and shows great promise for point-of-care and in-field detection of environmentally toxic mercury.     

Differential structural status of the RNA counterpart of an undecamer quasi-palindromic DNA sequence present in LCR of human β-globin gene cluster

Our previous work on structural polymorphism shown at a single nucleotide polymorphism (SNP) (A→G) site located on HS4 region of locus control region (LCR) of β-globin gene has established a hairpin→duplex equilibrium corresponding to A→B like DNA transition (Kaushik M, Kukreti, R., Grover, D., Brahmachari, S.K. and Kukreti S. Nucleic Acids Res. 2003; Kaushik M, Kukreti S. Nucleic Acids Res. 2006). The G-allele of A→G SNP has been shown to be significantly associated with the occurrence of β-thalassemia. Considering the significance of this 11-nt long quasi-palindromic sequence [5′-TGGGG(G/A)CCCCA; HP(G/A)11] of β-globin gene LCR, we further explored the differential behavior of the same DNA sequence with its RNA counterpart, using various biophysical and biochemical techniques. In contrast to its DNA counterpart exhibiting a A→B structural transition and an equilibrium between duplex and hairpin forms, the studied RNA oligonucleotide sequence [5′-UGGGG(G/A)CCCCA; RHP(G/A)11] existed only in duplex form (A-conformation) and did not form hairpin. The single residue difference from A to G led to the unusual thermal stability of the RNA structure formed by the studied sequence. Since, naturally occurring mutations and various SNP sites may stabilize or destabilize the local DNA/RNA secondary structures, these structural transitions may affect the gene expression by a change in the protein–DNA recognition patterns.     

Mutational analysis of adeno-associated virus type 2 Rep68 protein endonuclease activity on partially single-stranded substrates

The endonuclease activity of the Rep68 and Rep78 proteins (Rep68/78) of adeno-associated virus type 2 (AAV) cuts at the terminal resolution site (trs) within the hairpin structure formed by the AAV inverted terminal repeats. Recent studies suggest that a DNA unwinding function of Rep68/78 may be required for endonuclease activity. We demonstrate that several mutant proteins which are endonuclease negative on a fully duplex hairpin substrate are endonuclease positive on a partially single-stranded hairpin substrate. Truncation analysis revealed that the endonuclease function is contained within the first 200 amino acids of Rep68/78. This endonucleolytic cleavage is believed to involve the covalent attachment of Rep68/78 to the trs via a phosphate-tyrosine linkage. A previous report (S. L. Walker, R. S. Wonderling, and R. A. Owens, J. Virol. 71:2722-2730, 1997) suggested that tyrosine 152 was part of the active site. We individually mutated each tyrosine within the first 200 amino acids of the Rep68 moiety of a maltose binding protein-Rep68/78 fusion protein to phenylalanine. Only mutation of tyrosine 156 resulted in a protein incapable of covalent attachment to a partially single-stranded hairpin substrate, suggesting that tyrosine 156 is part of the endonuclease active site.     

纳米金标记核酸探针检测小反刍兽疫 病毒核酸的研究

试验旨在探讨利用纳米金标记寡核苷酸探针快速检测小反刍兽疫病毒核酸的方法。针对小反刍兽疫病毒N基因的高度保守区设计两条特异寡核苷酸探针,一条探针5’端修饰生物素,另一条探针3’端修饰巯基。巯基化的探针通过Au-S键连接到纳米金颗粒上。靶核酸两端分别与两条探针结合,形成"生物素化探针-靶核酸-纳米金探针"复合物,该复合物通过生物素-亲和素系统,固定在固相载体上,最后银染放大信号。通过肉眼观察法、光镜观察法、分光光度法分析银染灰度,从而间接测定靶核酸的量。初步检测PPRV核酸最低浓度达10fmol/L,所需时间约为1.5h。该方法灵敏度高、操作简单,为临床检测小反刍兽疫病毒核酸提供实验数据和技术支持。     

Simple colorimetric DNA detection based on hairpin assembly reaction and target-catalytic circuits for signal amplification

A simple strategy of colorimetric DNA detection is presented based on a hairpin assembly reaction and target-catalytic DNA circuits to achieve enzyme-free signal amplification. The method employed two hairpin species (H1 and H2), which were stable and unable to hybridize in the absence of target. In the presence of target, the target hybridized with hairpin H1 and the opened hairpin H1 hybridized with hairpin H2, allowing the target to be displaced. H1 and H2 were respectively attached to gold nanoparticles, allowing the duplex formed from H1 and H2 to be visualized with the naked eye. The displaced target again triggered the next round of strand exchange reaction to achieve signal amplification. The method may have a wide range of sensor applications because it is enzyme-free and simple to perform.     

Identification of nuclear proteins that specifically interact with adeno-associated virus type 2 inverted terminal repeat hairpin DNA.

A palindromic hairpin duplex containing the inverted terminal repeat sequence of adeno-associated virus type 2 (AAV) DNA was used as a substrate in gel retardation assays to detect putative proteins that specifically interact with the AAV hairpin DNA structures. Nuclear proteins were detected in extracts prepared from human KB cells coinfected with AAV and adenovirus type 2 that interacted with the hairpin duplex but not in nuclear extracts prepared from uninfected, AAV-infected, or adenovirus type 2-infected KB cells. The binding was specific for the hairpin duplex, since no binding occurred with a double-stranded DNA duplex with the identical nucleotide sequence. Furthermore, in competition experiments, the binding could be reduced with increasing concentrations of the hairpin duplex but not with the double-stranded duplex DNA with the identical nucleotide sequence. S1 nuclease assays revealed that the binding was sensitive to digestion with the enzyme, whereas the protein-bound hairpin duplex was resistant to digestion with S1 nuclease. The nucleotide sequence involved in the protein binding was localized within the inverted terminal repeat of the AAV genome by methylation interference assays. These nuclear proteins may be likely candidates for the pivotal enzyme nickase required for replication or resolution (or both) of single-stranded palindromic hairpin termini of the AAV genome.     

Fluorescence detection of thrombin using autocatalytic strand displacement cycle reaction and a dual-aptamer DNA sandwich assay

A sensitive and specific sandwich assay for the detection of thrombin is described. Two affiliative aptamers were used to increase the assay specificity through sandwich recognition. Recognition DNA loaded on gold nanoparticles (AuNPs) partially hybridized with the initiator DNA, which was displaced by surviving DNA. After the initiator DNA was released into the solution, one hairpin structure was opened, which in turn opened another hairpin structure. The initiator DNA was displaced and released into the solution again by another hairpin structure because of the hybridized reaction. Then the released initiator DNA initiated another autocatalytic strand displacement reaction. A sophisticated network of three such duplex formation cycles was designed to amplify the fluorescence signal. Other proteins, such as bovine serum albumin and lysozyme, did not interfere with the detection of thrombin. This approach enables rapid and specific thrombin detection with reduced costs and minimized material consumption compared with traditional assay processes. The detection limit of thrombin was as low as 4.3 × 10^?13 M based on the AuNP amplification and the autocatalytic strand displacement cycle reaction. This method could be used in biological samples with excellent selectivity.     

SinoDuplex: An Improved Duplex Sequencing Approach to Detect Low-frequency Variants in Plasma cfDNA Samples

Accurate detection of low frequency mutations from plasma cell-free DNA in blood using targeted next generation sequencing technology has shown promising benefits in clinical settings. Duplex sequencing technology is the most commonly used approach in liquid biopsies. Unique molecular identifiers are attached to each double-stranded DNA template, followed by production of low-error consensus sequences to detect low frequency variants. However, high sequencing costs have hindered application of this approach in clinical practice. Here, we have developed an improved duplex sequencing approach called Sino Duplex, which utilizes a pool of adapters containing pre-defined barcode sequences to generate far fewer barcode combinations than with random sequences, and implemented a novel computational analysis algorithm to generate duplex consensus sequences more precisely. Sino Duplex increased the output of duplex sequencing technology, making it more cost-effective. We evaluated our approach using reference standard samples and cell-free DNA samples from lung cancer patients. Our results showed that Sino Duplex has high sensitivity and specificity in detecting very low allele frequency mutations. The source code for Sino Duplex is freely available at https://github.com/Sin Oncology/sinoduplex.     

Modified Proofreading PCR for Detection of Point Mutations,Insertions and Deletions Using a ddNTP-Blocked Primer

The development of simple, accurate, rapid and cost-effective technologies for mutation detection is crucial to the early diagnosis and prevention of numerous genetic diseases, pharmacogenetics, and drug resistance. Proofreading PCR (PR-PCR) was developed for mutation detection in 1998 but is rarely applied due to its low efficiency in allele discrimination. Here we developed a modified PR-PCR method using a ddNTP-blocked primer and a mixture of DNA polymerases with and without the 3''-5'' proofreading function. The ddNTP-blocked primer exhibited the best blocking efficiency to avoid nonspecific primer extension while the mixture of a tiny amount of high-fidelity DNA polymerase with a routine amount of Taq DNA polymerase provided the best discrimination and amplification effects. The modified PR-PCR method is quite capable of detecting various mutation types, including point mutations and insertions/deletions (indels), and allows discrimination amplification when the mismatch is located within the last eight nucleotides from the 3''-end of the ddNTP-blocked primer. The modified PR-PCR has a sensitivity of 1-5 × 10² copies and a selectivity of 5 × 10^-5 mutant among 10⁷ copies of wild-type DNA. It showed a 100% accuracy rate in the detection of P72R germ-line mutation in the TP53 gene among 60 clinical blood samples, and a high potential to detect rifampin-resistant mutations at low frequency in Mycobacterium tuberculosis using an adaptor and a fusion-blocked primer. These results suggest that the modified PR-PCR technique is effective in detection of various mutations or polymorphisms as a simple, sensitive and promising approach.     

DNA-based biosensor for monitoring pH in vitro and in living cells

DNA is a promising material for the construction of a biosensor or bioindicator because its structure is sensitive to the binding of cofactors. In the current studies, we found that a combination of two DNA oligonucleotides, 5'-TCTTTCTCTTCT-3' and 5'-AGAAAGAGAAGA-3', exhibit a novel structural transition from a Watson-Crick antiparallel duplex to a parallel Hoogsteen duplex as the pH changes from pH 7.0 to 5.0. By labeling this DNA for fluorescence resonance energy transfer, we were able to develop a sensitive pH indicator that can detect changes between pH 7.0 and 5.0. Moreover, using DNA-based hairpin parallel-stranded duplex in conjunction with fluorescence microscopy, we were able to observe the pH changes in living cells during apoptosis as an easily detected change in color. These results indicate that the DNA-based pH indicator should be useful for detecting pH changes between pH 7.0 and 5.0 in living cells.     

Ultrasensitive signal-on DNA biosensor based on nicking endonuclease assisted electrochemistry signal amplification

Combining the advantages of signal-on strategy and nicking endonuclease assisted electrochemistry signal amplification (NEAESA), a new sensitive and signal-on electrochemical DNA biosensor for the sequence specific DNA detection based on NEAESA has been developed for the first time. A Hairpin-shape probe (HP), containing the target DNA recognition sequence, is thiol-modified at 5' end and immobilized on gold electrode via Au-S bonding. Subsequently, the HP modified electrode is hybridized with target DNA to form a duplex. Then the nicking endonuclease is added and nicks the HP strand in the duplex. After nicking, 3'-ferrocene (Fc)-labeled part complementary probe (Fc-PCP) is introduced on the electrode surface by hybridizing with the thiol-modified HP fragment, which results in the generation of electrochemical signal. Hence, the DNA biosensor is constructed successfully. The present DNA biosensor shows a wide linear range of 5.0×10(-13)-5.0×10(-8)M for detecting target DNA, with a low detection limit of 0.167pM. The proposed strategy does not require any amplifying labels (enzymes, DNAzymes, nanoparticles, etc.) for biorecognition events, which avoids false-positive results to occur frequently. Moreover, the strategy has the benefits of simple preparation, convenient operation, good selectivity, and high sensitivity. With the advantages mentioned above, this simple and sensitive strategy has the potential to be integrated in portable, low cost and simplified devices for diagnostic applications.     

DNA sexing of Nipponia nippon by duplex polymerase chain reaction

The aim of this study was to develop a rapid, simple, sensitive, and accurate duplex polymerase chain reaction (PCR) to sex Nipponia nippon, a monomorphic bird. Amplification by duplex PCR of a sex-related gene on the female chromosome and the 12S rRNA gene yielded good results using genomic DNA extracted from a feather follicle or the membranes of eggshell samples. To simplify the DNA extraction procedure, a simple boiling method was used. Our simple boiling DNA extraction method produced similar PCR amplification results as when using DNA extracted using TRIzol. Sex determination in the endangered Nipponia nippon is of crucial value to breeding programs. The duplex PCR protocol that we developed provides a simple sex identification method that is based on amplification of a sex-related gene, and we anticipate that it will facilitate effective conservation and management of Nipponia nippon.     

CuO thin film based uric acid biosensor with enhanced response characteristics

An electrochemical approach for detection of individual single nucleotide polymorphisms (SNPs) based on nucleobase-conjugated apoferritin probe loaded with metal phosphate nanoparticles is reported. Coupling of the nucleotide-modified nanoparticle probe to the mutant sites of duplex DNA was induced by DNA polymerase I (Klenow fragment) to preserve Watson-Crick base-pairing rules. After sequential liquid hybridization of biotinylated DNA probes with mutant DNA and complementary DNA, the resulting duplex DNA helixes were captured to the surface of magnetic beads through a well known and specific biotin-streptavidin affinity binding. For signaling each of eight possible Single-nucleotide polymorphisms (SNPs), Pb, Cu, Cd and Zn phosphate-loaded apoferritin nanoparticle probes were linked to adenosine (A), cytidine (C), guanosine (G), and thymidine (T) mononucleotides, respectively. Monobase-conjugated apoferritin probes were coupled to the mutant sites of the formed duplex DNA in the presence of DNA polymerase. Electrochemical stripping analyses of the metals loaded in apoferritin nanoparticle probes provide a means for detection and quantification of mutant DNA. Each mutation captures different nucleotide-conjugated apoferritin probe and provide a distinct four-potential voltammogram, whose peak potentials reflect the identity of the mismatch. The method is sensitive enough to accurately determine AG mutation, as the most thermodynamically stable mismatch to detect, in the range of 50-600 pM. The proposed protocol provides a simple, fast, cost-effective, accurate and sensitive method for detection of SNPs.     

CCR: a rapid and simple approach for mutation detection.

We describe a simple approach for detecting known mutations in genomic DNA. The strategy entails a DNA amplification reaction that combines the use of thermostable DNA polymerase and ligase, and that has been designated the Combined Chain Reaction (CCR). CCR consists of four phases: denaturation, annealing, elongation and ligation. Unlike most PCR-based mutation detection systems it relies on mismatch between primer and template at the primer 5'ends. It is rapid and simple, and requires neither the use of radioactivity, nor polyacrylamide gel electrophoresis, nor autoradiography for mutation detection at the single base-pair level.     

RNA mutations of prox1 detected in human esophageal cancer cells by the shifted termination assay

We have recently reported a novel finding that a candidate tumor suppressor gene prox1 suffered adenosine-to-inosine (A-to-I) RNA mutation without genomic mutation in a subset of human cancer cells and lost its function. Hence, screening of mutations in both cDNA and genomic DNA could be important in the analysis of causes for cancers. Here, we applied a sensitive, accurate, and simple method, called shifted termination assay (STA) for detection of an A-to-I RNA mutation (R334G) in prox1. We prepared PCR-amplified samples containing the target base of RNA mutation from cDNAs and genomic DNAs of various cell lines and clinical samples, to demonstrate that the STA method can be used to identify not only genomic mutations but also RNA mutations more effectively compared to sequencing. By means of STA, we found prox1 R334G RNA mutations but not genomic DNA mutations in 4 of 8 cases of esophageal cancers. This method can help us to detect RNA mutation effectively and progress research of a potential oncogenic principle.     

Lateral flow strip for visual detection of K-ras mutations based on allele-specific PCR

Objectives

To develop a convenient and sensitive point-of-care test for detecting gene mutations based on allele-specific PCR.

Results

To develop a lateral flow strip for visual detection of K-ras mutations based on a modified PCR, a specific DNA tag was covalently linked to the 5′-end of each primer by a nine-carbon linker to produce a sticky end. One of the sticky ends of the PCR products bound to gold nano-particles, while the other sticky end was captured onto a nitrocellulose membrane of lateral flow strips. The lateral flow strip showed a great sensitivity, which detected mutations in as low as 10 tumor cells. The positive rate and accuracy of the lateral flow strip for blood samples were over 92 and 96 %, respectively.

Conclusions

The lateral flow strip provides an easy method for sensitive detection of gene mutations based on allele specific-PCR.

     

Segregation of a single outboard left-end origin is essential for the viability of parvovirus minute virus of mice

During DNA replication, the hairpin telomeres of Minute Virus of Mice (MVM) are extended and copied to create imperfectly palindromic duplex junction sequences that bridge adjacent genomes in concatameric replicative-form DNA. These are resolved by the viral initiator protein, NS1, but mechanisms employed at the two telomeres differ. Left-end:left-end junctions are resolved asymmetrically at a single site, OriLTC, by NS1 acting in concert with a host factor, parvovirus initiation factor (PIF). Replication segregates doublet and triplet sequences, initially present as unpaired nucleotides in the bubble region of the left-end hairpin stem, to either side of the junction. These act as spacers between the NS1 and PIF binding sites, and their asymmetric distribution sets up active (OriLTC) and inactive (OriLGAA) forms of OriL. We used a reverse genetic approach to disrupt this asymmetry and found that neither opposing doublets nor triplets in the hairpin bubble were tolerated. Viable mutants were isolated at low frequency and found to contain second-site mutations that either restored the asymmetry or crippled one PIF binding site. These mutations either inactivated the inboard or activated the outboard form of OriL, a polarity that strongly suggests that, in the genus Parvovirus, an active inboard OriL is lethal.     

A novel multi-walled carbon nanotube-based antibody conjugate for quantitative and semi-quantitative lateral flow assays

In this study, the multi-walled carbon nanotubes (MWCNTs) were applied in lateral flow strips (LFS) for semi-quantitative and quantitative assays. Firstly, the solubility of MWCNTs was improved using various surfactants to enhance their biocompatibility for practical application. The dispersed MWCNTs were conjugated with the methamphetamine (MET) antibody in a non-covalent manner and then manufactured into the LFS for the quantitative detection of MET. The MWCNTs-based lateral flow assay (MWCNTs-LFA) exhibited an excellent linear relationship between the values of test line and MET when its concentration ranges from 62.5 to 1500 ng/mL. The sensitivity of the LFS was evaluated by conjugating MWCNTs with HCG antibody and the MWCNTs conjugated method is 10 times more sensitive than the one conjugated with classical colloidal gold nanoparticles. Taken together, our data demonstrate that MWCNTs-LFA is a more sensitive and reliable assay for semi-quantitative and quantitative detection which can be used in forensic analysis.     

Discriminating duplex and hairpin oligonucleotides using chemical shifts: application to the anticodon stem-loop of Escherichia coli tRNA(Phe)

A sensitive NMR spectroscopic method for detection of duplex forms of self-complementary nucleic acid sequences has been implemented. The G.U wobble base pair formed between a (15)N-labeled strand and an unlabeled probe strand is used to identify the duplex. The guanine imino resonance, with its characteristic chemical shift, is detected using a 2D (15)N-(1)H heteronuclear multiple quantum coherence (HMQC) spectrum and provides a sensitive and unambiguous route to hairpin-duplex discrimination. The method has been used to identify the duplex and hairpin forms of an RNA oligonucleotide at concentrations of approximately 20 microM. This method has also been used to rule out possible duplex formation of an RNA oligonucleotide corresponding to the unmodified anticodon stem-loop of Escherichia coli tRNA(Phe) and suggests that this hairpin has a 3 nt loop.