DNA and protein microarray printing on silicon nitride waveguide surfaces

Sputtered silicon nitride optical waveguide surfaces were silanized and modified with a hetero-bifunctional crosslinker to facilitate thiol-reactive immobilization of contact-printed DNA probe oligonucleotides, streptavidin and murine anti-human interleukin-1 beta capture agents in microarray formats. X-ray photoelectron spectroscopy (XPS) was used to characterize each reaction sequence on the native silicon oxynitride surface. Thiol-terminated DNA probe oligonucleotides exhibited substantially higher surface printing immobilization and target hybridization efficiencies than non-thiolated DNA probe oligonucleotides: strong fluorescence signals from target DNA hybridization supported successful DNA oligonucleotide probe microarray fabrication and specific capture bioactivity. Analogously printed arrays of thiolated streptavidin and non-thiolated streptavidin did not exhibit noticeable differences in either surface immobilization or analyte capture assay signals. Non-thiolated anti-human interleukin-1 beta printed on modified silicon nitride surfaces reactive to thiol chemistry exhibited comparable performance for capturing human interleukin-1 beta analyte to commercial amine-reactive microarraying polymer surfaces in sandwich immunoassays, indicating substantial non-specific antibody-surface capture responsible for analyte capture signal.     

Spatial Organization of the Chicken α-Globin Gene Domain in Cells of Different Origins

Hybridization with an oligonucleotide array was used to map the regions of DNA anchorage to the nuclear matrix. Matrix-associated DNA served as a hybridization probe. To obtain the oligonucleotide array, 60-mer oligonucleotides regularly distributed throughout the genome region of interest at 2-kb intervals were immobilized on a nylon filter. The organization of DNA into loop domains was studied in a 100-kb region of chicken chromosome 16, including the α -globin gene cluster. A 40-kb DNA loop, which was fixed to the nuclear matrix and harbored all α-globin genes, was observed in erythroid cells. One of its anchorage regions colocalized with matrix associated region (MAR) and an insulator found previously in the 5′ region of the chicken α-globin gene domain. The spatial (domain-loop) organization of the α-globin gene cluster in lymphoid cells proved to be strikingly different from that in erythroid cells.     

Oligonucleotide-primed in situ DNA synthesis (PRINS): a method for chromosome mapping, banding, and investigation of sequence organization

Oligonucleotides were annealed to complementary sequences in fixed human metaphase chromosomes and extended with DNA polymerase. The newly synthesized fragments were labeled by incorporating bio-11-dUTP instead of TTP, and the sites of synthesis were detected by immunocytochemistry, using fluorochromes as the reporter molecules. We have obtained clear localization with oligonucleotides from alphoid (centromeric sequences), simple sequence (satellite) DNAs, a variety of Alu-dispersed repeated sequences, and oligonucleotides derived from the Tetrahymena and Trypanosoma telomere-specific sequences. The simple sequence and alphoid oligonucleotides gave results at least comparable to those obtained using the whole molecule as a probe for in situ hybridization, whereas the Alu oligonucleotides produced a diversity of results which depended on the absolute length and location of the oligonucleotide within the Alu sequence. The telomere-specific oligomers also produced a variety of results. The G-rich Trypanosoma oligomer and its complementary C-rich sequence produced strong telomeric signals and some interstitial signals on mouse chromosomes, but only weak telomeric signals on human chromosomes. The G-rich Tetrahymena oligomer produced detectable telomeric signals on human chromosomes. The technique appears to be a valuable extension of present tools for mapping and examining the organization of DNA sequences within chromosomes.     

Rapid identification of clones using the same degenerate oligonucleotide mixture for both screening and sequencing

A simple and rapid strategy for distinguishing between positively hybridizing colonies and false positive-hybridization signals is described. The isolation of a specific DNA sequence depends on the ability to distinguish between a clone that contains the correct sequence and a false hybridization-positive or background signal. This procedure utilizes the same oligonucleotide mixture both as a screening probe and as a sequencing primer. The mixture of oligonucleotides is used as a primer to obtain sequence information directly from double-stranded DNA. Conditions for sequencing with oligonucleotides having up to 64-fold degeneracy are described. Since the sequence information obtained is directly adjacent to the site of oligonucleotide:DNA hybridization, it is necessary to know only a minimal length of DNA or peptide sequence to both design oligonucleotide probes and confirm the authenticity of the hybridization positives. The advantages of the degenerate oligonucleotide sequencing method include the rapid, reliable identification of authentic versus false hybridization positives made directly without subcloning into single-stranded M13 phage, without sequencing large regions of DNA, or without synthesizing sequence-specific primers.     

Liquid-based hybridization assay with real-time detection in miniaturized array platforms

An assay for the fluorescent detection of short oligonucleotide probe hybridization in miniaturized high-density array platforms is presented. It combines hybridization in solution with real-time fluorescent detection, which involves measurement of fluorescence increase by means of an induced fluorescence resonance energy transfer. The feasibility of this approach using DNA or RNA as a target, and short DNA- as well as LNA (locked nucleic acid)-modified oligonucleotides as probes is shown. The presented approach could potentially contribute to a significant increase in the throughput of large-scale genomic applications, such as oligofingerprinting and genotyping, and also reduce material consumption.     

Potato flour viscosity improvement is associated with the expression of a wheat LMW-glutenin gene

It has been previously shown that expression of a high-molecular-weight glutenin (HMW-GS) in transgenic wheat seeds resulted in the improvement of flour functional properties. In this study, potato flour viscosity was improved through a specific expression of a low-molecular-weight glutenin (LMW-GS-MB1) gene in tuber. The resulting construct was introduced into potato leaf explants (Solanum tuberosum cv Kennebec) through Agrobacterium tumefaciens-mediated gene transfer. Southern and Northern analysis of transgenic potato confirmed that the integration of LMW-GS-MB1 in genomic DNA was stable and its mRNA was abundant in transgenic line 16 tubers. Western blot analysis of line 16 extract shows a LMW-GS subunit accumulation in tuber. To demonstrate the capacity of transgenic lines to produce tubers with improved flour functional properties, transgenic lines 9 and 16 exhibiting, respectively, moderate and high expression of LMW-GS-MB1 mRNA and nontransgenic plants were transferred to field plots. The mean viscosity value of flour obtained from the field-grown tubers of transgenic line 16 exhibited a 3-fold increase in viscosity at 23 degrees C when compared to flour from nontransgenic tubers.     

Probing distance and electrical potential within a protein pore with tethered DNA

DNA molecules tethered inside a protein pore can be used as a tool to probe distance and electrical potential. The approach and its limitations were tested with alpha-hemolysin, a pore of known structure. A single oligonucleotide was attached to an engineered cysteine to allow the binding of complementary DNA strands inside the wide internal cavity of the extramembranous domain of the pore. The reversible binding of individual oligonucleotides produced transient current blockades in single channel current recordings. To probe the internal structure of the pore, oligonucleotides with 5' overhangs of deoxyadenosines and deoxythymidines up to nine bases in length were used. The characteristics of the blockades produced by the oligonucleotides indicated that single-stranded overhangs of increasing length first approach and then thread into the transmembrane beta-barrel. The distance from the point at which the DNA was attached and the internal entrance to the barrel is 43 A, consistent with the lengths of the DNA probes and the signals produced by them. In addition, the tethered DNAs were used to probe the electrical potential within the protein pore. Binding events of oligonucleotides with an overhang of five bases or more, which threaded into the beta-barrel, exhibited shorter residence times at higher applied potentials. This finding is consistent with the idea that the main potential drop is across the alpha-hemolysin transmembrane beta-barrel, rather than the entire length of the lumen of the pore. It therefore explains why the kinetics and thermodynamics of formation of short duplexes within the extramembranous cavity of the pore are similar to those measured in solution, and bolsters the idea that a "DNA nanopore" provides a useful means for examining duplex formation at the single molecule level.     

Expression of Potato Virus Y Coat Protein Gene in Transgenic Potato

Potato virus Y (PVY) N coat protein (CP) coding sequence was cloned into a plant expression vector pMON316 under the CaMV 35S promoter. Leaf discs of potato (Solanum tuberosum) were used to Agrobacterium-mediated gene transfer. A large number of regenerated putative transgenic plants were obtained based on kanamycin resistance. Using total DNA purified from transgenic plants as templates and two oligonucleotides synthesized from 5' and 3' of the PVY coat protein gene as primers, the authors carried out polymerase chain reaction (PCR) to check the presence of this gene and obtained a 0. 8 kb specific DNA fragment after 35 cycles of amplification. Southern blot indicated that the PCR product was indeed PVY CP gene which had been integrated into the potato genome. Enzyme-linked immunosorbent assay (ELISA) of our transgenic plants showed that CP gene was expressed in at least some transgenic potato plants.     

Engineered selective plant male sterility through pollen‐specific expression of the EcoRI restriction endonuclease

Unintended gene flow from transgenic plants via pollen, seed and vegetative propagation is a regulatory concern because of potential admixture in food and crop systems, as well as hybridization and introgression to wild and weedy relatives. Bioconfinement of transgenic pollen would help address some of these concerns and enable transgenic plant production for several crops where gene flow is an issue. Here, we demonstrate the expression of the restriction endonuclease EcoRI under the control of the tomato pollen‐specific LAT52 promoter is an effective method for generating selective male sterility in Nicotiana tabacum (tobacco). Of nine transgenic events recovered, four events had very high bioconfinement with tightly controlled EcoRI expression in pollen and negligible‐to‐no expression other plant tissues. Transgenic plants had normal morphology wherein vegetative growth and reproductivity were similar to nontransgenic controls. In glasshouse experiments, transgenic lines were hand‐crossed to both male‐sterile and emasculated nontransgenic tobacco varieties. Progeny analysis of 16 000–40 000 seeds per transgenic line demonstrated five lines approached (>99.7%) or attained 100% bioconfinement for one or more generations. Bioconfinement was again demonstrated at or near 100% under field conditions where four transgenic lines were grown in close proximity to male‐sterile tobacco, and 900–2100 seeds per male‐sterile line were analysed for transgenes. Based upon these results, we conclude EcoRI‐driven selective male sterility holds practical potential as a safe and reliable transgene bioconfinement strategy. Given the mechanism of male sterility, this method could be applicable to any plant species.     

Evaluating oligonucleotide properties for DNA microarray probe design

Most current microarray oligonucleotide probe design strategies are based on probe design factors (PDFs), which include probe hybridization free energy (PHFE), probe minimum folding energy (PMFE), dimer score, hairpin score, homology score and complexity score. The impact of these PDFs on probe performance was evaluated using four sets of microarray comparative genome hybridization (aCGH) data, which included two array manufacturing methods and the genomes of two species. Since most of the hybridizing DNA is equimolar in CGH data, such data are ideal for testing the general hybridization properties of almost all candidate oligonucleotides. In all our data sets, PDFs related to probe secondary structure (PMFE, hairpin score and dimer score) are the most significant factors linearly correlated with probe hybridization intensities. PHFE, homology and complexity score are correlating significantly with probe specificities, but in a non-linear fashion. We developed a new PDF, pseudo probe binding energy (PPBE), by iteratively fitting dinucleotide positional weights and dinucleotide stacking energies until the average residue sum of squares for the model was minimized. PPBE showed a better correlation with probe sensitivity and a better specificity than all other PDFs, although training data are required to construct a PPBE model prior to designing new oligonucleotide probes. The physical properties that are measured by PPBE are as yet unknown but include a platform-dependent component. A practical way to use these PDFs for probe design is to set cutoff thresholds to filter out bad quality probes. Programs and correlation parameters from this study are freely available to facilitate the design of DNA microarray oligonucleotide probes.     

植物病毒检测芯片的杂交条件优化

利用芯片点样仪将5种侵染马铃薯的病毒/类病毒(苜蓿花叶病毒、黄瓜花叶病毒、黄瓜花叶病毒-卫星病毒、马铃薯病毒Y、马铃薯块茎纺锤状类病毒)的保守区寡核苷酸(Oligonucleotide,oligo)探针和PCR探针点样于玻片,并以植物18S rRNA作为内参照制成基因芯片。研究探针浓度、杂交时间、杂交温度以及点样液对芯片杂交的影响,并验证优化后病毒检测芯片的特异性。结果表明,寡核苷酸探针浓度介于5-20 ?mol/L之间对杂交信号强度影响不大,PCR探针浓度与杂交信号强度间呈线性关系;在45℃杂交4 h时,芯片的杂交信号最强,且该条件下进行杂交对两种探针芯片的影响趋势一致;点样液中以DMSO的杂交效果最好。经过整体条件优化后的两种探针芯片在杂交检测上具有较高的特异性,适于检测植物病毒。     

Application of synthetic oligonucleotides to the diagnosis of human genetic diseases

Under appropriate conditions synthetic oligonucleotide hybridization probes display essentially absolute hybridization specificity. That is, every nucleotide must form a Watson-Crick base pair in order that the probe forms a stable duplex. All of the non-Watson-Crick base pairs, including G-T, have a destabilizing effect. Thus, it is possible to choose stringent conditions of hybridization such that, while a perfectly matched duplex between an oligonucleotide and complementary DNA will form, duplexes mismatched at one or more position will not. Mutations in a single base in the DNA sequence of a gene can and do result in genetic diseases. The hybridization of oligonucleotides to the region of DNA containing these base changes would be affected by the mutations and thus, oligonucleotide hybridization provides a means of detecting single base changes. In an attempt to develop a non-radioactive method for the detection of human genetic diseases, we have prepared biotinylated-oligonucleotides by an enzymatic method. An oligonucleotide probe (23-mer) containing a single biotinylated deoxyuridine residue at the 3'-terminus was prepared by a primer extention reaction using E. coli DNA polymerase I (Klenow fragment). The probe could be specifically and tightly bound with Avidin D in 1 M NaCl. It could be hybridized to a plasmid DNA containing a perfectly matched complementary sequence, but not to a DNA containing 5 non-consecutive non-complementary bases. The hybridized biotinylated probe could be visualized by Avidin D and biotinylated alkaline phosphatase, even when 1.8 ng of the plasmid DNA (0.5 fmol) was used. A general approach to the enzymatic synthesis of oligonucleotides containing a single biotinylated deoxyuridine at the 3' end is described.     

Oligonucleotide array for identification and detection of pythium species

A DNA array containing 172 oligonucleotides complementary to specific diagnostic regions of internal transcribed spacers (ITS) of more than 100 species was developed for identification and detection of Pythium species. All of the species studied, with the exception of Pythium ostracodes, exhibited a positive hybridization reaction with at least one corresponding species-specific oligonucleotide. Hybridization patterns were distinct for each species. The array hybridization patterns included cluster-specific oligonucleotides that facilitated the recognition of species, including new ones, belonging to groups such as those producing filamentous or globose sporangia. BLAST analyses against 500 publicly available Pythium sequences in GenBank confirmed that species-specific oligonucleotides were unique to all of the available strains of each species, of which there were numerous economically important ones. GenBank entries of newly described species that are not putative synonyms showed no homology to sequences of the spotted species-specific oligonucleotides, but most new species did match some of the cluster-specific oligonucleotides. Further verification of the specificity of the DNA array was done with 50 additional Pythium isolates obtained by soil dilution plating. The hybridization patterns obtained were consistent with the identification of these isolates based on morphology and ITS sequence analyses. In another blind test, total DNA of the same soil samples was amplified and hybridized on the array, and the results were compared to those of 130 Pythium isolates obtained by soil dilution plating and root baiting. The 13 species detected by the DNA array corresponded to the isolates obtained by a combination of soil dilution plating and baiting, except for one new species that was not represented on the array. We conclude that the reported DNA array is a reliable tool for identification and detection of the majority of Pythium species in environmental samples. Simultaneous detection and identification of multiple species of soilborne pathogens such as Pythium species could be a major step forward for epidemiological and ecological studies.     

Spread of recombinant DNA by roots and pollen of transgenic potato plants,identified by highly specific biomonitoring using natural transformation of an Acinetobacter sp

Transgenic potato plants with the nptII gene coding for neomycin phosphotransferase (kanamycin resistance) as a selection marker were examined for the spread of recombinant DNA into the environment. We used the recombinant fusion of nptII with the tg4 terminator for a novel biomonitoring technique. This depended on natural transformation of Acinetobacter sp. strain BD413 cells having in their genomes a terminally truncated nptII gene (nptII'; kanamycin sensitivity) followed by the tg4 terminator. Integration of the recombinant fusion DNA by homologous recombination in nptII' and tg4 restored nptII, leading to kanamycin-resistant transformants. DNA of the transgenic potato was detectable with high sensitivity, while no transformants were obtained with the DNA of other transgenic plants harboring nptII in different genetic contexts. The recombinant DNA was frequently found in rhizosphere extracts of transgenic potato plants from field plots. In a series of field plot and greenhouse experiments we identified two sources of this DNA: spread by roots during plant growth and by pollen during flowering. Both sources also contributed to the spread of the transgene into the rhizospheres of nontransgenic plants in the vicinity. The longest persistence of transforming DNA in field soil was observed with soil from a potato field in 1997 sampled in the following year in April and then stored moist at 4 degrees C in the dark for 4 years prior to extract preparation and transformation. In this study natural transformation is used as a reliable laboratory technique to detect recombinant DNA but is not used for monitoring horizontal gene transfer in the environment.     

Oligonucleotide-priming methods for the chromosome-specific labelling of alpha satellite DNA in situ

It is demonstrated that either general staining of the centromeric regions of all primate chromosomes, or selective staining of the centromeric region of specific chromosomes, may be obtained in preparations of metaphase chromosomes by probing specifically for different regions within the alpha satellite DNA monomer. In order to exploit observed patterns of sequence variation within the monomer for this purpose, we have developed two new DNA analysis methods. In PRimed IN Situ labelling (PRINS), synthetic oligonucleotides derived from subsections of the monomer are hybridized to the chromosomes. The oligonucleotides then serve as primers for the in situ incorporation of biotin-labelled nucleotides catalysed by Klenow polymerase. Incorporated biotin is visualized with fluorescein isothiocyanate-labelled avidin (FITC-avidin). In Primed Amplification Labelling (PAL), biotin-labelled hybridization probes are produced in a polymerase chain reaction (PCR, Saiki et al. 1985), in which two synthetic oligonucleotide primers anneal within the same monomer. With the right choice of primers libraries of labelled probes derived from most monomers present as templates are produced. If DNA from a specific chromosome is used as template, then the resulting probe mixture gives stronger and more chromosome-specific signals in in situ hybridization experiments than does a cloned alpha satellite DNA probe derived from the same chromosome. The results obtained indicate that the alpha-repeat monomer is composed of regions with different degrees of chromosome specificity.