DNA Hybridization Chain Reaction Research Articles

Combinatorial analysis of mRNA expression patterns in mouse embryos using hybridization chain reaction.

Multiplexed fluorescent hybridization chain reaction (HCR) and advanced imaging techniques can be used to evaluate combinatorial gene expression patterns in whole mouse embryos with unprecedented spatial resolution. Using HCR, DNA probes complementary to mRNA targets trigger chain reactions in which metastable fluorophore-labeled DNA HCR hairpins self-assemble into tethered fluorescent amplification polymers. Each target mRNA is detected by a probe set containing one or more DNA probes, with each probe carrying two HCR initiators. For multiplexed experiments, probe sets for different target mRNAs carry orthogonal initiators that trigger orthogonal DNA HCR amplification cascades labeled by spectrally distinct fluorophores. As a result, in situ amplification is performed for all targets simultaneously, and the duration of the experiment is independent of the number of target mRNAs. We have used multiplexed fluorescent in situ HCR and advanced imaging technologies to address questions of cell heterogeneity and tissue complexity in craniofacial patterning and anterior neural development. In the sample protocol presented here, we detect three different mRNA targets: Tg(egfp), encoding the enhanced green fluorescent protein (GFP) transgene (typically used as a control); Twist1, encoding a transcription factor involved in cell lineage determination and differentiation; and Pax2, encoding a transcription factor expressed in the mid-hindbrain region of the mouse embryo.

A novel colorimetric sensor for Hg(2+) based on hybridization chain reaction and silver nanowire amplification.

Through the silver ion catalysis to form colored KMnO4, and combined with the DNA hybridization chain reaction and silver nanowire for signal amplification, a highly sensitive and selective colorimetric sensor has been developed for the detection of Hg(2+).

A simple strategy for signal amplification based on DNA hybridization chain reaction for thrombin detection

Multiple enzymes were introduced onto the surface of electrode, which resulted in significant signal amplification.

Next-generation in situ hybridization chain reaction: higher gain, lower cost, greater durability.

Hybridization chain reaction (HCR) provides multiplexed, isothermal, enzyme-free, molecular signal amplification in diverse settings. Within intact vertebrate embryos, where signal-to-background is at a premium, HCR in situ amplification enables simultaneous mapping of multiple target mRNAs, addressing a longstanding challenge in the biological sciences. With this approach, RNA probes complementary to mRNA targets trigger chain reactions in which metastable fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The properties of HCR lead to straightforward multiplexing, deep sample penetration, high signal-to-background, and sharp subcellular signal localization within fixed whole-mount zebrafish embryos, a standard model system for the study of vertebrate development. However, RNA reagents are expensive and vulnerable to enzymatic degradation. Moreover, the stringent hybridization conditions used to destabilize nonspecific hairpin binding also reduce the energetic driving force for HCR polymerization, creating a trade-off between minimization of background and maximization of signal. Here, we eliminate this trade-off by demonstrating that low background levels can be achieved using permissive in situ amplification conditions (0% formamide, room temperature) and engineer next-generation DNA HCR amplifiers that maximize the free energy benefit per polymerization step while preserving the kinetic trapping property that underlies conditional polymerization, dramatically increasing signal gain, reducing reagent cost, and improving reagent durability.

Antimicrobial resistance and antimicrobial resistance genes in marine bacteria from salmon aquaculture and non‐aquaculture sites

Antimicrobial resistance (AR) detected by disc diffusion and antimicrobial resistance genes detected by DNA hybridization and polymerase chain reaction with amplicon sequencing were studied in 124 marine bacterial isolates from a Chilean salmon aquaculture site and 76 from a site without aquaculture 8 km distant. Resistance to one or more antimicrobials was present in 81% of the isolates regardless of site. Resistance to tetracycline was most commonly encoded by tetA and tetG; to trimethoprim, by dfrA1, dfrA5 and dfrA12; to sulfamethizole, by sul1 and sul2; to amoxicillin, by blaTEM ; and to streptomycin, by strA-strB. Integron integrase intl1 was detected in 14 sul1-positive isolates, associated with aad9 gene cassettes in two from the aquaculture site. intl2 Integrase was only detected in three dfrA1-positive isolates from the aquaculture site and was not associated with gene cassettes in any. Of nine isolates tested for conjugation, two from the aquaculture site transferred AR determinants to Escherichia coli. High levels of AR in marine sediments from aquaculture and non-aquaculture sites suggest that dispersion of the large amounts of antimicrobials used in Chilean salmon aquaculture has created selective pressure in areas of the marine environment far removed from the initial site of use of these agents.

Hybridization Chain Reaction Amplification of MicroRNA Detection with a Tetrahedral DNA Nanostructure-Based Electrochemical Biosensor

There remains a great challenge in the sensitive detection of microRNA because of the short length and low abundance of microRNAs in cells. Here, we have demonstrated an ultrasensitive detection platform for microRNA by combining the tetrahedral DNA nanostructure probes and hybridization chain reaction (HCR) amplification. The detection limits for DNA and microRNA are 100 aM and 10 aM (corresponding to 600 microRNAs in a 100 μL sample), respectively. Compared to the widely used supersandwich amplification, the detection limits are improved by 3 orders of magnitude. The uncontrolled surface immobilization and consumption of target molecules that limit the amplification efficiency of supersandwich are eliminated in our platform. Taking advantage of DNA nanotechnology, we employed three-dimensional tetrahedral DNA nanostructure as the scaffold to immobilize DNA recognition probes to increase the reactivity and accessibility, while DNA nanowire tentacles are used for efficient signal amplification by capturing multiple catalytic enzymes in a highly ordered way. The synergetic effect of DNA tetrahedron and nanowire tentacles have proven to greatly improve sensitivity for both DNA and microRNA detection.

Label-free technology for the amplified detection of microRNA based on the allosteric hairpin DNA switch and hybridization chain reaction

By using the allosteric hairpin DNA switch, a novel assay for the detection of microRNA (miRNA) let-7a via a hybridization chain reaction (HCR) was introduced. Briefly, the hairpin DNA switch probe is a single-stranded DNA consisting of a streptavidin (SA) aptamer sequence, a target binding sequence and a certain sequence that acts as a trigger of the HCR. In the presence of target let-7a, the hairpin DNA switch would open and expose the stem region sequences, where a part of this sequence acts as initiator sequence strands for the HCR and triggers a cascade of hybridization events that yields nicked double helices analogous to alternating copolymers, another part is the SA aptamer sequence which activates its binding affinity to SA on SA-coated magnetic particles. The hybridization event could be sensitively detected via an instantaneous derivatization reaction between a special chemiluminescence (CL) reagent, 3,4,5-trimethoxylphenylglyoxal (TMPG) and the guanine nucleotides within the target, the hairpin DNA switch probe, and HCR helices to form an unstable CL intermediate for the generation of light. Our results show that the coupling of the hairpin DNA switch probe and the HCR for the amplified detection of let-7a achieves a better performance (e.g. wide linear response range: 0.1-1000 fmol, low detection limit: 0.1 fmol, and high specificity). Furthermore, this approach could be easily applied to the detection of let-7a in human lung cells, and extended to detect other types of miRNA and proteins such as PDGF based on aptamers. We believe such advancements will represent a significant step towards improved diagnostics and more personalized medical treatment.

Using dual-polarization interferometry to study surface-initiated DNA hybridization chain reactions in real time

In this study we used dual-polarization interferometry to investigate DNA hybridization chain reactions (HCRs) at solid–liquid interfaces. We monitored the effects of variations in mass, thickness, and density of the immobilized initiator on the subsequent HCRs at various salt concentrations. At low salt concentrations, the single-stranded DNA (ssDNA) initiator was attached uniformly to the chip surface. At high salt concentrations, it lay on the surface at the onset of the immobilization process, but the approaching ssDNA forced the pre-immobilized ssDNA strands to extend into solution as a result of increased electrostatic repulsion between the pre-adsorbed and approaching ssDNA chains. Injection of a mixture of H1 and H2 increased the mass and thickness of the films initially, but thereafter the thickness decreased. These changes indicate that the long double-stranded DNA that formed lay on the surface, rather than extended into the solution, thereby suppressing the subsequent initiation activity of the released single-strand parts of H1 and H2. Increasing the salt concentration increased the HCR efficiency and reaction rate. The HCR efficiency of the initiator ssDNA immobilized on its 5′ end was higher than that immobilized on its 3′ end, suggesting that the released single-strand parts of H1 and H2 close to the chip surface decreased the initiation activity relative to those of the ones extending into solution.

DNA branch migration reactions through photocontrollable toehold formation.

Strand displacement cascades are commonly used to make dynamically assembled structures. Particularly, the concept of "toehold-mediated DNA branch migration reactions" has attracted considerable attention in relation to dynamic DNA nanostructures. However, it is a challenge to obtain and control the formation of pure 1:1 ratio DNA duplexes with toehold structures. Here, for the first time, we report a photocontrolled toehold formation method, which is based on the photocleavage of 2-nitrobenzyl linker-embedded DNA hairpin precursor structures. UV light irradiation (λ ≈ 365 nm) of solutions containing these DNA hairpin structures causes the complete cleavage of the nitrobenzyl linker, and pure 1:1 DNA duplexes with toehold structures are easily formed. Our experimental results indicate that the amount of toehold can be controlled by simply changing the dose of UV irradiation and that the resulting toehold structures can be used for subsequent toehold-mediated DNA branch migration reactions, e.g., DNA hybridization chain reactions. This newly established method will find broad application in the construction of light-powered, controllable, and dynamic DNA nanostructures or large-scale DNA circuits.

In Situ Hybridization Chain Reaction Amplification for Universal and Highly Sensitive Electrochemiluminescent Detection of DNA

In this work, we describe a new universal and highly sensitive strategy for electrochemiluminescent (ECL) detection of sequence specific DNA at the femtomolar level via in situ hybridization chain reaction (HCR) signal amplification. The DNA capture probes are self-assembled on a gold electrode. The presence of the target DNA and two hairpin helper DNAs leads to the formation of extended dsDNA polymers through HCR on the electrode surface. The in situ, HCR-generated dsDNA polymers cause the intercalation of numerous ECL indicators (Ru(phen)(3)(2+)) into the dsDNA grooves, resulting in significantly amplified ECL signal output. The proposed strategy combines the amplification power of the DNA HCR and the inherent high sensitivity of the ECL technique and enables low femtomolar detection of sequence specific DNA. The developed strategy also shows high selectivity against single-base mismatch sequences, which makes our new universal and highly sensitive HCR-based method a useful addition to the amplified DNA detection arena.

Quartz Crystal Microbalance Studies on Surface-Initiated DNA Hybridization Chain Reaction

The hybridization chain reaction (HCR), a type of nucleic acid amplification method, was studied by a quartz crystal resonator technique. We probed the properties of the boundary DNA layer with a thickness of the viscous penetration depth at the crystal-solution interface. According to the relationship between the change of resonant frequency and the mass adsorbed on the crystal surface, the kinetics of surface-initiated HCR was measured, as well as the viscosity and the shear modulus of H1/H2 solution with different concentrations near a solid substrate. This work provides a new method for study- ing nucleic acid reactions at solid surface and mechanistic insight for HCR-based amplification and biodetection. Keywords hybridization chain reaction; quartz crystal microbalance; DNA; viscosity; interface; penetration depth

DNA Base Pair Hybridization and Water-Mediated Metastable Structures Studied by Molecular Dynamics Simulations

The base pair hybridization of a DNA segment was studied using molecular dynamics simulation. The results show the obvious correlation between the probability of successful hybridization and the accessible surface area to water of two successive base pairs, including the unpaired base pair adjacent to paired base pair and this adjacent paired base pair. Importantly, two metastable structures in an A-T base pair were discovered by the analysis of the free energy landscape. Both structures involved addition of a water molecule to the linkage between the two nucleobases in one base pair. The existence of the metastable structures provide potential barriers to the Watson-Crick base pair, and numerical simulations show that those potential barriers can be surmounted by thermal fluctuations at higher temperatures. These studies contribute an important step toward the understanding of the mechanism in DNA hybridization, particularly the effect of temperature on DNA hybridization and polymerase chain reaction. These observations are expected to be helpful for facilitating experimental bio/nanotechnology designs involving fast hybridization.

Rapid Detection by Genomic Amplification of Loofah Witches’ Broom Phytoplasma in Leafhopper Vectors

AbstractLoofah (Luffa cylindrical) is a common vegetable crop in Taiwan and other Asian countries. Reported here is a novel rapid approach for detecting loofah witches’ broom (LfWB) phytoplasma in single leafhoppers. Field samples of suspected diseased plants and potential vectors from southern Taiwan were processed to test for the presence of the LfWB phytoplasmas using both strain‐specific DNA hybridization (DH) and polymerase chain reaction (PCR) assays. The commonest pathogen causing loofah disease in southern Taiwan is LfWB phytoplasma. Leafhoopers collected at nine locations near LfWB‐infected plants were found to be positive for LfWB by PCR / DH at an incidence of 28.5–40.0%. Of the different leafhopper species tested, only Hishimonus concavus was positive for LfWB, suggesting that H. concavus is a natural vector of LfWB in Taiwan. Using our proposed primers in this PCR assay, a single LfWB‐infected leafhopper can be detected rapidly and directly.

Pepper golden mosaic virus Affecting Tomato Crops in the Baja California Peninsula, Mexico.

In the state of Baja California Sur, tomatoes (Lycopersicon esculentum Mill.) are cultivated on approximately 1,000 ha. Occurrence of viral diseases is currently causing low yields and severe losses. Virus-like symptoms (severe leaf curling, stunting, reduced leaf size, and mosaic patterns) were observed on 99% of tomato plants in 2002 in La Paz, Baja California Sur. Whiteflies (Bemisia tabaci Gennadius) were present in affected fields and appeared to be associated with the disease. The virus was experimentally transmitted from infected plants to tomato and peppers seedlings by whiteflies and as infectious DNA (replicative form) by mechanical and biolistic inoculation. Symptoms similar to those found in the field were observed in experimental transmission assays. DNA from inoculated plants was extracted and analyzed by DNA hybridization and polymerase chain reaction (PCR) using degenerate (1) and specific (2) primers. The PCR products (1.1 kb) obtained from the common region (GenBank Accession No. AY368336) suggested the presence of a bipartite geminivirus. The nucleotide sequence of the PCR products showed a 98% identity to Pepper golden mosaic virus-Tamaulipas strain (PepGMV-Tam) in the intergenic region (IR). Similar identities (97%) were obtained by using the predicted amino acid sequences of the amino termini of the coat proteins. Identities in the replication proteins (92%) and IR iterative sequence analyses show that the PepGMV-La Paz isolate is a closely related strain of PepGMV-Tam. To our knowledge, this is the first report of PepGMV affecting tomato crops in Baja California Sur, Mexico.

Isolation and association of Escherichia coli AIDA-I/STb, rather than EAST1 pathotype, with diarrhea in piglets and antibiotic sensitivity of isolates.

To identify emerging Escherichia coli that have the potential to cause diarrhea in pigs, the prevalence of E. coli pathotypes was determined among 170 and 120 isolates from diarrheic and nondiarrheic piglets, respectively. The isolates were tested for F4, F5, F6, F18, and F41 fimbriae, for E. coli attaching and effacing (EAE), porcine attaching and effacing-associated (Paa), and adhesin involved in diffuse adherence (AIDA-I) factors, for LT, STa, STb, and enteroaggregative heat-stable (EAST1) enterotoxins, and for Shiga toxins (Stxl, Stx2, and Stx2e), using DNA hybridization and polymerase chain reaction. All isolates were O-serotyped and tested for antibiotic resistance against 10 drugs. Seventeen different pathotypes, accounting for 40.0% of the isolates, were recovered from diarrheic piglets. The main pathotypes included EAST1 (13.5%), F4/LT/STb/EAST1 (6.5%), AIDA-I/STb/EAST1 (4.1%), F5/STa (2.9%), EAE/EAST1 (2.9%), and AIDA-I/F18 (2.3%). Only 3 pathotypes, EAE (11.7%), EAST1 (10.8%), and EAE/EAST1 (3.3%), were recovered from nondiarrheic piglets. Paa factor was detected in 8.8% and 7.5% of isolates from diarrheic and nondiarrheic piglets, respectively, and always was associated with other virulence determinants. Overall, 22.9% of isolates from diarrheic piglets appeared to be enteropathogens: enterotoxigenic E. coli (11.7%), enteropathogenic E. coli (3.5%), and E. coli isolates (3.0%) for which none of the above adherence factors was detected. Pathotypes AIDA-I/STb/EAST1 and AIDA-I/STb were isolated only from diarrheic piglets and accounted for 4.7% of isolates. Strains of these pathotypes induced diarrhea when inoculated into newborn colostrum-deprived pigs, in contrast to an isolate positive only for EAST1, which did not induce diarrhea. Antibiotic sensitivity test showed that isolates of the AIDA-I/STb/EAST1 and AIDA-I/STb pathotypes were the only strains sensitive to enrofloxacin, gentamicin, neomycin, and trimethoprim-sulfamethoxazole. This study showed that at least 20.5% of isolates from diarrheic piglets appeared to be associated with AIDA-I/STb pathotype and that EAST1 pathotype is probably not an important marker for diarrhea in piglets.

Frequency and Seasonal Distribution of Pear Psylla Infected with the Pear Decline Phytoplasma in California Pear Orchards

ABSTRACT Pear decline (PD) is an important disease of Pyrus communis fruiting cultivars in Europe, Asia, and the Americas. PD is caused by a phloem-limited phytoplasma that, in California, is transmitted from diseased to healthy trees by pear psylla, Cacopsylla pyricola. The percentage of phytoplasma-infected pear psylla has never been assessed in the United States in field-collected insects. Pear psylla were collected monthly from PD-infected trees from three orchards in northern California. Individual psylla were tested for the presence of PD phytoplasma, using both a quantitative DNA hybridization and PD phytoplasma-specific polymerase chain reaction (PCR) assays. The percentage of infected psylla ranged from 0 to 45% depending on the orchard, the month and year collected, and the method of detection. The PD phytoplasma was detected in both the winterform and summerform pear psylla. Significantly more infected psylla were detected with PCR than with DNA hybridization analysis in two of the three orchards. The number of PD phytoplasma per pear psylla was estimated to range from 1 x 10(6) to 8.2 x 10(7). The percentage of PD-infected pear psylla found in the three northern California pear orchards suggests that both winterform and summerform pear psylla could be important in the transmission of PD.

Identification of Phytoplasma Taxa and Insect Vectors of Peach Yellow Leaf Roll Disease in California.

Two peach diseases in California, western X-disease (WX) and peach yellow leaf roll (PYLR) are caused by two genetically distinct phytoplasmas. Based on symptoms alone, WX and PYLR cannot be reliably distinguished. The objectives of this study were to determine which phytoplasma was causing PYLR disease in peach orchards planted near pear orchards and which phytoplasmas were present in potential insect vectors captured in both peach and pear orchards. Leafhoppers and pear psylla were collected from yellow sticky traps placed in three pear orchards and four peach orchards located in the Sacramento Valley from 1994 to 1996. DNA was extracted from potential vectors and suspect diseased trees, and analyzed for the presence of the WX and PYLR phytoplasmas using strain-specific DNA hybridization or polymerase chain reaction (PCR) assays. The most abundant phytoplasma detected in diseased peach trees was the PYLR phytoplasma. In 1994 and 1995, 10 to 25% of groups of pear psylla tested positive for the PYLR phytoplasma, while no psylla groups tested positive for the WX phytoplasma. Only one captured leafhopper tested positive for the WX phytoplasma. These results indicate the pear psylla is the primary vector of PYLR in northern California.

Speciation and distribution of the members of the Anopheles punctulatus (Diptera: Culicidae) group in Papua New Guinea.

Mosquito collections were made throughout the mainland of Papua New Guinea to identify the members of the Anopheles punctulatus group present and to determine their distribution. Identification was made using morphology, DNA hybridization, and polymerase chain reaction (PCR)-RFLP analysis. Nine members of the group were identified: An. farauti s.s. Laveran, An. farauti 2, An. koliensis Owen, and An. punctulatus Dönitz, were common and widespread; An. farauti 4 was restricted to the north of the central ranges where it was common; An. farauti 6 was found only in the highlands above 1,000 m; and An. farauti 3, An. sp. near punctulatus and An. clowi Rozeboom & Knight were uncommon and had restricted distributions. Identification of An. koliensis and An. punctulatus using proboscis morphology was found to be unreliable wherever An. farauti 4 occurred. The distribution and dispersal of the members of the An. punctulatus group is discussed in regard to climate, larval habitats, distance from the coast, elevation, and proximity to human habitation.

A homogeneous europium cryptate-based assay for the diagnosis of mutations by time-resolved fluorescence resonance energy transfer.

Oligonucleotide ligation assay (OLA) is considered to be a very useful methodology for the detection and characterization of mutations, particularly for clinical purposes. The fluorescence resonance energy transfer between a fluorescent donor and a suitable fluorophore as acceptor has been applied in the past to several scientific fields. This technique is well adapted to nucleic acid analysis such as DNA sequencing, DNA hybridization and polymerase chain reaction. We describe here a homogeneous format based on the use of a rare earth cryptate label as donor: tris-bipyridine-Eu(3+). The long-lived fluorescence of this label makes it possible to reach a high sensitivity by using a time-resolved detection mode. A non-radiative energy transfer technology, known as time-resolved amplification of cryptate emission (TRACE((R))) characterized by a temporal and spectral selectivity has been developed. The TRACE((R)) detection of characterized single nucleotide polymorphism using the OLA for allelic discrimination is proposed. We demonstrate the potentialities of this OLA-TRACE((R)) methodology through the analysis of K-ras oncogene point mutations.

Hybridization analysis of the gene encoding a hemolysin (suilysin) of Streptococcus suis type 2: evidence for the absence of the gene in some isolates

A hemolysin gene was cloned from a virulent strain of Streptococcus suis type 2 strain 1933. Analysis of the gene and its product revealed that it is identical to a previously reported hemolysin (suilysin) of S. suis type 2. Southern hybridization analysis of the digested total genomic DNA from S. suis with the cloned hemolysin DNA sequences as probe indicated that the hemolysin gene is present as a single copy on the genome. Genomic DNA of 63 isolates of S. suis encompassing all known serotypes were examined by DNA hybridization and polymerase chain reaction (PCR) studies for the presence of the hemolysin gene homolog. The results of both techniques were identical and demonstrated the absence of the hemolysin gene in some isolates. In DNA hybridization studies, three DNA probes derived from the hemolysin encoding gene were used. Results showed that sequences encoding the C-terminal 257 amino acid residues (Probe 1) were the most conserved and hybridized to a 1.2 kb fragment in 32 (51%) strains and a 4.0 kb fragment in 23 (36%) strains respectively. Thus, Probe 2 hybridized to the DNA of 55 (87%) of the isolates tested. The first probe (Probe 1) comprising almost the entire hemolysin gene and the third probe (Probe 3) which consisted of the N-terminal sequences hybridized only to a 4.0 kb fragment in 23 (36%) of the strains tested. Eight (13%) of the strains tested were hybridization and PCR negative. The hybridization of the C-terminal end sequences (Probe 2) to the 1.2 kb fragment in 32 (51%) of the strains and the lack of hybridization of the probes to eight (13%) strains may suggest the presence of different types of hemolysin molecule in S. suis strains.