Conventional Hybridization Research Articles

Comparative Genomic Hybridization (CGH) for Prenatal Diagnosis of Wolf-Hirschhorn Syndrome

Wolf-Hirschhorn Syndrome (WHS) is a rare genetic condition caused by partial deletion of the short arm of chromosome 4 (4p-). Variability in 4p deletions and rearrangements leads to a wide spectrum of clinical manifestations of this disease. Most prenatal WHS diagnoses are associated with large 4p deletions identified by conventional cytogenetic techniques; however some submicroscopic deletions can only be diagnosed using molecular techniques. In this case report, a combined diagnostic approach based on conventional karyotyping and comparative genomic hybridization (CGH) offered a quick and definitive result to allow accurate prognoses and genetic counseling of WHS for the family.

Alternative applications for distinct RNA sequencing strategies.

Recent advances in RNA library preparation methods, platform accessibility and cost efficiency have allowed high-throughput RNA sequencing (RNAseq) to replace conventional hybridization microarray platforms as the method of choice for mRNA profiling and transcriptome analyses. RNAseq is a powerful technique to profile both long and short RNA expression, and the depth of information gained from distinct RNAseq methods is striking and facilitates discovery. In addition to expression analysis, distinct RNAseq approaches also allow investigators the ability to assess transcriptional elongation, DNA variance and exogenous RNA content. Here we review the current state of the art in transcriptome sequencing and address epigenetic regulation, quantification of transcription activation, RNAseq output and a diverse set of applications for RNAseq data. We detail how RNAseq can be used to identify allele-specific expression, single-nucleotide polymorphisms and somatic mutations and discuss the benefits and limitations of using RNAseq to monitor DNA characteristics. Moreover, we highlight the power of combining RNA- and DNAseq methods for genomic analysis. In summary, RNAseq provides the opportunity to gain greater insight into transcriptional regulation and output than simply miRNA and mRNA profiling.

A simple and cost-effective molecular diagnostic system and DNA probes synthesized by light emitting diode photolithography

This work introduces a novel chip to be used in the future as a simple and cost-effective method for creating DNA arrays using light emission diode (LED) photolithography. The DNA chip platform contains 24 independent reaction sites, which allows for the testing of a corresponding amount of patients’ samples in hospital. An array of commercial UV LEDs and lens systems was combined with a microfluidic flow system to provide patterning of 24 individual reaction sites, each with 64 independent probes. Using the LED array instead of conventional laser exposure systems or micro-mirror systems significantly reduces the cost of equipment. The microfluidic system together with microfluidic flow cells drastically reduces the amount of used reagents, which is important due to the high cost of commercial reagents. The DNA synthesis efficiency was verified by fluorescence labeling and conventional hybridization.

In situ hybridization of superparamagnetic iron-biomolecule nanoparticles.

The increase in interest in the integration of organic-inorganic nanostructures in recent years has promoted the use of hybrid nanoparticles (HNPs) in medicine, energy conversion, and other applications. Conventional hybridization methods are, however, often long, complicated, and multistepped, and they involve biomolecules and discrete nanostructures as separate entities, all of which hinder the practical use of the resulting HNPs. Here, we present a novel, in situ approach to synthesizing size-specific HNPs using Fe-biomolecule complexes as the building blocks. We choose an anticancer peptide (p53p, MW 1.8 kDa) and an enzyme (GOx, MW 160 kDa) as model molecules to demonstrate the versatility of the method toward different types of molecules over a large size range. We show that electrostatic interaction for complex formation of metal hydroxide ion with the partially charged side of biomolecule in the solution is the key to hybridization of metal-biomolecule materials. Electrochemical deposition is then used to produce hybrid NPs from these complexes. These HNPs with controllable sizes ranging from 30 nm to 3.5 μm are found to exhibit superparamagnetic behavior, which is a big challenge for particles in this size regime. As an example of greatly improved properties and functionality of the new hybrid material, in vitro toxicity assessment of Fe-GOx HNPs shows no adverse effect, and the Fe-p53p HNPs are found to selectively bind to cancer cells. The superparamagnetic nature of these HNPs (superparamagnetic even above the size regime of 15-20 nm!), their biocompatibility, and the direct integration approach are fundamentally important to biomineralization and general synthesis strategy for bioinspired functional materials.

Assistant deoxyribonucleic acid recycling with Zn2+ and molecular beacon for electrochemical detection of deoxyribonucleic acid via target-triggered assembly of mutated DNAzyme

A novel enzyme-free amplification strategy was designed for sensitive electrochemical detection of deoxyribonucleic acid (DNA) based on Zn2+ assistant DNA recycling via target-triggered assembly of mutated DNAzyme. A gold electrode was used to immobilize molecular beacon (MB) as the recognition probe and perform the amplification procedure. In the presence of target DNA, the hairpin probe 1 was opened, and the DNAzyme was liberated from the caged structure. The activated DNAzyme first hybridized and then cleaved the MB in the presence of cofactor Zn2+. After cleavage, the MB was cleaved into two pieces and the ferrocene (Fc) labeled piece dissociated from the gold electrode, thus obviously decreasing the Fc signal and forming a free DNAzyme strand. Finally, each target-induced activated DNAzyme underwent many cycles to trigger the cleavage of many MB substrates. Therefore, the peak current of Fc dramatically decreased to approximately zero. The strategy showed a detection limit at 35fM levels, which was about 2 orders of magnitude lower than that of the conventional hybridization without Zn2+-based amplification. The Zn2+ assistant DNA recycling offers a versatile platform for DNA detection in a cost-effective manner, and has a promising application in clinical diagnosis.

RNA recognition by a human antibody against brain cytoplasmic 200 RNA.

Diverse functional RNAs participate in a wide range of cellular processes. The RNA structure is critical for function, either on its own or as a complex form with proteins and other ligands. Therefore, analysis of the RNA conformation in cells is essential for understanding their functional mechanisms. However, no appropriate methods have been established as yet. Here, we developed an efficient strategy for panning and affinity maturation of anti-RNA human monoclonal antibodies from a naïve antigen binding fragment (Fab) combinatorial phage library. Brain cytoplasmic 200 (BC200) RNA, which is also highly expressed in some tumors, was used as an RNA antigen. We identified MabBC200-A3 as the optimal binding antibody. Mutagenesis and SELEX experiments showed that the antibody recognized a domain of BC200 in a structure- and sequence-dependent manner. Various breast cancer cell lines were further examined for BC200 RNA expression using conventional hybridization and immunoanalysis with MabBC200-A3 to see whether the antibody specifically recognizes BC200 RNA among the total purified RNAs. The amounts of antibody-recognizable BC200 RNA were consistent with hybridization signals among the cell lines. Furthermore, the antibody was able to discriminate BC200 RNA from other RNAs, supporting the utility of this antibody as a specific RNA structure-recognizing probe. Intriguingly, however, when permeabilized cells were subjected to immunoanalysis instead of purified total RNA, the amount of antibody-recognizable RNA was not correlated with the cellular level of BC200 RNA, indicating that BC200 RNA exists as two distinct forms (antibody-recognizable and nonrecognizable) in breast cancer cells and that their distribution depends on the cell type. Our results clearly demonstrate that anti-RNA antibodies provide an effective novel tool for detecting and analyzing RNA conformation.

Vaccine strain varicella-zoster virus–induced central nervous system vasculopathy as the presenting feature of DOCK8 deficiency

Vaccine strain varicella-zoster virus–induced central nervous system vasculopathy as the presenting feature of DOCK8 deficiency

Highly improved specificity for hybridization-based microRNA detection by controlled surface dissociation

Poor specificity has been a lingering problem in many microRNA profiling methods, particularly surface hybridization-based methods such as microarrays. Here, we carefully investigated surface hybridization and dissociation processes of a number of sequentially similar microRNAs against nucleic acid capture probes. Single-base mismatched microRNAs were similarly hybridized to a complementary DNA capture probe and thereby poorly discriminated during conventional stringent hybridization. Interestingly, however, mismatched microRNAs showed significantly faster dissociation from the probe than the perfectly matched microRNA. Systematic analysis of various washing conditions clearly demonstrated that extremely high specificity can be obtained by releasing non-specific microRNAs from assay surfaces during a stringent and controlled dissociation step. For instance, compared with stringent hybridization, surface dissociation control provided up to 6-fold better specificity for Let-7a detection than for other Let-7 family microRNAs. In addition, a synthetically introduced single-base mismatch on miR206 was almost completely discriminated by optimized surface dissociation of captured microRNAs, while this mismatch was barely distinguished from target miR206 during stringent hybridization. Furthermore, a single dissociation condition was successfully used to simultaneously measure four different microRNAs with extremely high specificity using melting temperature-equalized capture probes. The present study on selective dissociation of surface bound microRNAs can be easily applied to various hybridization based detection methods for improved specificity.

Chromosomal aberrations in primary PDGFRA-mutated gastrointestinal stromal tumors

Approximately 15% of gastrointestinal stromal tumors (GISTs) harbor mutations in the platelet-derived growth factor receptor α (PDGFRA) gene. Chromosomal aberrations play a crucial role in tumor progression and correlate with clinical behavior. Imbalances, particularly in PDGFRA-mutated GISTs, have not yet been evaluated in larger series. We analyzed 53 PDGFRA-mutated GISTs (including 2 with corresponding metastases) for chromosomal imbalances by conventional comparative genomic hybridization and compared them with a historical collective of 122 KIT-mutated GISTs. PDGFRA exon 18 mutations (91% of cases) and exon 12 mutations (9% of cases) correlated significantly with gastric and intestinal sites, respectively. The most common aberrations were identical to those found in KIT-mutated GISTs, with -14q in 70%, -1p in 28%, and -22q in 17% of cases. Overall, there were significantly fewer chromosomal aberrations compared with KIT-mutated GISTs, with a mean of 2.8 (0.6 gains, 2.1 losses) aberrations per tumor. There was a statistically significant association of more than 5 chromosomal imbalances with intermediate/high-risk categories. Regarding specific chromosomal aberrations, -9p, -13q, and -22q correlated with intermediate/high risk, and -1p and +8q with poorer survival, although progression occurred in only 2 cases. Altogether, PDGFRA-mutated GISTs display the same chromosomal aberrations as KIT-mutated GISTs, although they have a lower degree of chromosomal instability in line with their generally favorable outcome.

Advances in Molecular Technologies and Platforms for the Diagnosis of Infectious Diseases

nfectious microbial pathogens constitute the largest cause of morbidity and mortality worldwide. Early diagnosis and rapid infection control measures can lead to improved outcomes, earlier discharges and reduced nosocomial infections. Conventional diagnostic methods for infectious diseases such as microscopy, culture, and immunological methods, in most cases, are not universally applicable, less sensitive and could take from days to months to complete depending on the pathogen. Molecular assays based on nucleic acids such as polymerase chain reaction (PCR) have improved the sensitivity, specificity and turn-around time in diagnostic microbiology laboratories. These tests are particularly important to detect very low levels of pathogens in clinical samples, and for organisms that have long half-lives or are non-culturable. However, individual molecular tests are available for only a limited number of the more common infectious agents. Moreover, infectious disease events arising from novel pathogens or genetic variants have significantly increased, recently, for which, routine diagnostic methods are not yet available. Therefore, molecular methods and technologies capable of detecting multiple pathogens in a single test have become available over the last few years. Although, these methods are based on the conventional nucleic acid amplification and hybridization chemistry, enhanced multiplexing capability has been achieved through innovations in nucleic acid labeling techniques, and post-amplification analytic methods and instrumentation. The availability of these test kits brought a new level of convenience to the physicians ordering practices, and to the laboratory personnel, as they require very little hands on time. However, these tests are yet unaffordable to many laboratories, and in many cases, the sensitivity is poor compared to that of single-target, real-time PCR assays. Looking into the future, the revolutionary, next generation sequencing (NGS) technology is now being considered as a potential method for rapid identification of hundreds of pathogens, in an unbiased manner, with a single test that could significantly benefit patients who are critically ill with undiagnosed disease.

Strategies for increasing the yield potential of cereals: case of rice as an example

AbstractRice is the most important food crop. Worldwide, more than 3.5 billion people depend upon rice for more than 20% of their calories. Global rice demand is estimated to rise from 676 million tons in 2010 to 763 million tons in 2020 and to further increase to 852 million tons in 2035. This is an overall increase of 176 million tons in the next 25 years. To meet this challenge rice production on existing land must be increased. Grain production is largely influenced by the yield potential of rice varieties. Therefore, improvement in the yield potential of rice is the major strategy to increase world rice production. Various strategies to increase the yield potential include; (1) conventional hybridization and selection, (2) ideotype breeding, (3) hybrid breeding, (4) exploitation of wild species germplasm, (5) enhancement of photosynthesis, (6) genomic approaches and (7) physiological approaches. Rice has become a model plant for genetics and breeding research. Advances in molecular biology and genomics, proteomics and metabolics have opened new avenues to apply innovative approaches to rice breeding. Molecular‐assisted selection (MAS) has become an integral component of germplasm improvement. A large number of genes/QTLs for various traits have been tagged with molecular markers to apply MAS for trait improvement. Genome sequence data have become an important source for detecting allelic variation. It is now possible to precisely understand the role of epigenetic changes in gene expression, thus understanding the stability of various stresses under changing environments. All these advances when integrated with conventional breeding will result in designer rice varieties to meet the challenges of rice food security in 21th century.

Application of Exonuclease III-Aided Target Recycling in Flow Cytometry: DNA Detection Sensitivity Enhanced by Orders of Magnitude

DNA-functionalized microspheres in conjugation with flow cytometry detection are widely used for high-throughput nucleic acid assays. Although such assays are rapid and capable of simultaneous analysis of multiple nucleic acid analytes in a single test, the intrinsic limitation in sensitivity remains challenging. Here we report a simple, highly sensitive, and reproducible method based on Exonuclease III-aided target recycling technique applied for DNA quantification in flow cytometry. By loading a high density of Cy5-labeled probe DNA on microspheres (15 μm), we achieved hitherto unreported DNA detection limit of 3.2 pM in flow cytometry bead assay, enhancing the sensitivity by a factor of over 56.8 compared to the conventional direct hybridization bead assay. Furthermore, we evaluated multiplexing capability by simultaneous detections of two target DNAs with FAM and Cy5 reporter conjugated probes. Therefore, the novel Exonuclease III-amplified flow cytometry bead assay has great potential for the rapid, sensitive, and accurate detection and quantification of nucleic acids in clinical diagnosis and biomedical research.

Nanoscale Superstructures Assembled by Polymerase Chain Reaction (PCR): Programmable Construction, Structural Diversity, and Emerging Applications

Polymerase chain reaction (PCR) is an essential tool in biotechnology laboratories and is becoming increasingly important in other areas of research. Extensive data obtained over the last 12 years has shown that the combination of PCR with nanoscale dispersions can resolve issues in the preparation DNA-based materials that include both inorganic and organic nanoscale components. Unlike conventional DNA hybridization and antibody-antigen complexes, PCR provides a new, effective assembly platform that both increases the yield of DNA-based nanomaterials and allows researchers to program and control assembly with predesigned parameters including those assisted and automated by computers. As a result, this method allows researchers to optimize to the combinatorial selection of the DNA strands for their nanoparticle conjugates. We have developed a PCR approach for producing various nanoscale assemblies including organic motifs such as small molecules, macromolecules, and inorganic building blocks, such as nanorods (NRs), metal, semiconductor, and magnetic nanoparticles (NPs). We start with a nanoscale primer and then modify that building block using the automated steps of PCR-based assembly including initialization, denaturation, annealing, extension, final elongation, and final hold. The intermediate steps of denaturation, annealing, and extension are cyclic, and we use computer control so that the assembled superstructures reach their predetermined complexity. The structures assembled using a small number of PCR cycles show a lower polydispersity than similar discrete structures obtained by direct hybridization between the nanoscale building blocks. Using different building blocks, we assembled the following structural motifs by PCR: (1) discrete nanostructures (NP dimers, NP multimers including trimers, pyramids, tetramers or hexamers, etc.), (2) branched NP superstructures and heterochains, (3) NP satellite-like superstructures, (4) Y-shaped nanostructures and DNA networks, (5) protein-DNA co-assembly structures, and (6) DNA block copolymers including trimers and pentamers. These results affirm that this method can produce a variety of chemical structures and in yields that are tunable. Using PCR-based preparation of DNA-bridged nanostructures, we can program the assembly of the nanoscale blocks through the adjustment of the primer intensity on the assembled units, the number of PCR cycles, or both. The resulting structures are highly complex and diverse and have interesting dynamics and collective properties. Potential applications of these materials include chirooptical materials, probe fabrication, and environmental and biomedical sensors.

Genetic improvement of Citrus for disease resistance

Progress in the genetic improvement of Citrus species was reviewed. Tools used for the genetic improvement of Citrus were categorised as conventional (introduction, selection and hybridisation) and non-conventional methods (mutation, somatic cell hybridisation and genetic engineering) of improvement. Genes linked with the disease resistance were characterised and tagged through molecular marker techniques such as Sequenced Characterised Amplified Region and Cleaved Amplified Polymorphic Sequences. Disease resistance genes showed both monogenic and polygenic inheritance. Conventional methods for disease resistance improvement of Citrus were bottleneck due to inadequate and lengthy breeding procedures. However, non-conventional methods, such as mutation breeding and protoplast fusion, have been routinely utilised for the production of disease resistant germplasm while novel genes from variable sources were used to transform Citrus species to induce resistance against diseases. These non-conventional techniques have been shown to overcome the disadvantages of conventional breeding procedures and could be regarded as rapid methods of genetic improvement as well as helpful to overcome the interspecies barrier.

The cDNA cloning of a novel bacterial blight-resistance gene <italic>ME137</italic>

Bacterial blight (BB) of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most serious diseases of rice worldwide [1]. Use of resistant varieties has become the most economic and effective method to control the disease. To date, more than 30 genes have been found and characterized. Five dominant R genes, Xa1, Xa3, Xa21, Xa26, Xa27, and two recessive R genes, Xa5, Xa13, have been cloned [2–8]. However, because of the differentiation of physiological race and the continually emerging of new toxic bacterial flora, cultivated varieties with narrower resistance spectrum will lose their resistance ability to BB in a short period [9]. Therefore, excavating and identification of new resistance genes are of significance to control BB. Wild rice species have long been used as genetic resources in rice breeding. Some genes that confer resistance to blight disease have been identified in wild rice. Xa21, which was first reported by Khush et al. [10], was the first gene found in wild rice (Oryza longistaminata). After cloning and application in breeding resistant varieties, resistance genes were identified successively in O. rufipogon, O. minuta, O. officinalis, and O. australiensis [11– 14]. A previous study revealed that some of O. meyeriana materials are immune to the BB disease [15]. So far the genome of O. meyeriana has not been demonstrated, thus it is difficult to investigate the resistance gene. Furthermore, because hybridization could not happen normally between O. meyeriana (GG genome 2n 1⁄4 24) and cultivated rice (AA genome), it is hard to use the resistance gene of O. meyeriana by conventional hybridization approaches for improving cultivated rice. All of these indicate that we should try some other approaches to find and identify novel disease-resistance genes. It is interesting why O. meyeriana can be immune to BB disease and what kind of genes is related to BB disease resistance in O. meyeriana. In this study, a novel BB resistance-related gene, named temporarily ME137, was cloned from O. meyeriana. This study would lay the foundation for finding, identification and usage of new resistance genes, and breeding resistant varieties. The main materials used in this study were as follows: X. oryzae pv. oryzae C1, O. meyeriana planted in a vinyl house (Biotechnology & Genetic Resources Institute, Yunnan Academy of Agricultural Sciences, Kunming, China) at natural temperature and light, RNAiso Plus (TaKaRa, Dalian, China), M-MLV-reverse transcriptase (TIANGEN, Beijing, China), 50-Full RACE kit (TaKaRa), SanPrep DNA gel extraction kit (Sangon, Shanghai, China), and pMD18-T Vector (TaKaRa). O. meyeriana was inoculated with C1 by the leafclipping method in the flowering stage. Fresh leaves inoculated were cut and collected for cloning cDNA. The fresh leaves at 1 d before Xoo induction and 1, 2, 3, 4, 5 d after Xoo induction were used for semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. All the materials were frozen in liquid nitrogen immediately after collection and stored at 2808C for further use. Total RNA was extracted from collected leaf tissues and amplified into first-strand cDNA. The Expressed Sequence Tag sequence that has low homology with previously cloned ones to obtain a novel resistance gene was chosen from suppression subtractive hybridization (SSH) library and confirmed by a pair of primers 137-1 (50-GACCCTGCC CAACA-30) and 137-2 (50-ACCGCTGCCTGAT-30). The SSH library was constructed by the conventional method, after O. meyeriana was inoculated with Xoo as tester, and non-inoculated materials as driver [16]. The primers used for amplification of actin gene were actin-F (50-GTATGG TGAAGGCTGGATTTGC-30) and actin-R (50-ATTTCCCG CTCTGCTGTTGTGG-30). To obtain 30 end of ME137, the primer AP-dT (50-CG CGGATCCAAGCTTATCGATTTTTTTTTTTTTTTTTT-30) instead of Oligo dT was used when amplified into first-strand cDNA. Then, a pair of primers AP (50-CGCGGATCCAA GCTTATCGA-30) and 137-1 (50-GACCCTGCCCAACA-30) were designed to get the 30 end of cDNA sequence. 50 RACE was performed as recommended by the manufactures. To obtain specific band, nested PCR was used. In the outer PCR, a pair of primers outer-F (50-CATGGCTACATGCTG Acta Biochim Biophys Sin 2013, 45: 422–424 | a The Author 2013. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmt029. Advance Access Publication 4 April 2013

DNA meter: Energy tunable, quantitative hybridization assay

We describe a novel hybridization assay that employs a unique class of energy tunable, bulge loop-containing competitor strands (C*) that hybridize to a probe strand (P). Such initial "pre-binding" of a probe strand modulates its effective "availability" for hybridizing to a target site (T). More generally, the assay described here is based on competitive binding equilibria for a common probe strand (P) between such tunable competitor strands (C*) and a target strand (T). We demonstrate that loop variable, energy tunable families of C*P complexes exhibit enhanced discrimination between targets and mismatched targets, thereby reducing false positives/negatives. We refer to a C*P complex between a C* competitor single strand and the probe strand as a "tuning fork," since the C* strand exhibits branch points (forks) at the duplex-bulge interfaces within the complex. By varying the loop to create families of such "tuning forks," one can construct C*P "energy ladders" capable of resolving small differences within the target that may be of biological/functional consequence. The methodology further allows quantification of target strand concentrations, a determination heretofore not readily available by conventional hybridization assays. The dual ability of this tunable assay to discriminate and quantitate targets provides the basis for developing a technology we refer to as a "DNA Meter." Here we present data that establish proof-of-principle for an in solution version of such a DNA Meter. We envision future applications of this tunable assay that incorporate surface bound/spatially resolved DNA arrays to yield enhanced discrimination and sensitivity.

Meiosis and sexual fertility of autotetraploid clones of greater yam Dioscorea alata L.

Meiosis in the tetraploid (2n = 80) males of Dioscorea alata L. was investigated for the first time. During metaphase I, the chromosomes were associated mostly in 6–8 quadrivalents and the remaining ones as bivalents with no trivalents or univalents. Anaphase I and subsequent stages of meiosis were normal. The observation of quadrivalents in the tetraploids provides cytological evidence for autotetraploidy. The autotetraploid males and females were highly fertile and they produced viable seeds on artificial pollination. Pollination between diploids (2n = 40) and tetraploids (2n = 80) were also successful via embryo rescue, producing triploid (2n = 60) progenies. The discovery of fertile autotetraploids could initiate polyploidy breeding in D. alata by conventional hybridization for the first time. The reduced number of quadrivalents and the high fertility of the autotetraploids are thought to be the result of partial diploidization of meiosis. The findings also refute the assumption of the allopolyploid origin of D. alata from two putative progenitors.

Effectiveness of three antimicrobial mouthrinses on the disinfection of toothbrushes stored in closed containers: a randomized clinical investigation by DNA Checkerboard and Culture

The aim of this in vivo study was to evaluate the efficacy of three antimicrobial solutions on the disinfection of toothbrushes after storage in closed containers. Sixteen healthy subjects were enrolled in this randomized cross-over clinical investigation. The study was conducted in four phases, in which mouthrinses (chlorhexidine gluconate-based or cetilpiridinium-based) and sterile tap water (control group) were used to individually store used toothbrushes in closed containers during 7 days of toothbrushing. Five toothbrushes were used as negative control for bacterial colonisation before contact with oral cavity. Conventional culture and DNA Checkerboard hybridization were used to detect bacterial contamination on the toothbrushes. Subsequently, the number of bacterial species on the bristles was estimated by the DNA Checkerboard method. One toothbrush presented bacterial contamination in the negative control test. Both culture and DNA Checkerboard showed positive signals of bacterial contamination in the toothbrushes with no differences in the frequency of detection. The control group showed higher total bacterial counts when compared with the mouthrinse groups. Porphyromonas gingivalis had the highest bacterial count followed by Parvimonas micra. Culture and DNA Checkerboard showed positive signals of bacterial contamination. Mouthrinses that contains 0.12% of chlorhexidine gluconate were more effective in reducing bacterial colonisation on the toothbrushes.

First Fruiting Intergeneric Hybrids between Citrus and Citropsis

Fruiting hybrids are reported for the first time between the genera Citrus L. and Citropsis (Engl.) Swing. & M.Kell. Conventional hybridization using the recently described species Citrus wakonai P.I.Forst. & M.W.Sm. and Citropsis gabunensis (Engl.) Swing. & M.Kell. resulted in high rates of fruit set and seed formation. Although seed were only half normal size, over 90% germinated without the need for embryo rescue techniques. Plant losses were high during the first few months but after six months, the 327 surviving hybrids were potted on. These grew vigorously on their own roots and 35 of them flowered within two years of sowing. Plants flowered continuously but all were pollen-sterile and ovaries abscised shortly after petal fall. However, at 25 months, two newly flowering hybrids began setting fruit. The development, identification, morphology, breeding efficiency, and future implications of this unique germplasm are described.

Energy-Tunable Quantitative Hybridization Assay

We describe here a novel molecular design approach to optimizing the sensitivity and selectivity of probe-target interactions. The methodology employs a novel hybridization assay which uses a unique class of energy tunable competitor strands (C∗) that hybridize to a probe strand (P). The assay is based on competitive binding equilibria for a common probe strand (P) between such competitor strands (C∗) and a target strand (T). We demonstrate that families of tunable C∗P complexes exhibit enhanced discrimination between targets and mismatched targets, thereby reducing false positives/negatives. The methodology also allows quantification of target strand concentrations, a determination heretofore not readily available by conventional hybridization assays. We present solution data that establish proof-of-principle for this energy-tunable quantitative hybridization assay. It is envisioned that future practical applications of this technology will be based on surface bound/spatially resolved DNA arrays.