Specific DNA Hybridization Research Articles

Gold-nanoparticle-based miniaturized laser-induced fluorescence probe for specific DNAhybridization detection: studies on size-dependent optical properties

A compact, highly specific, inexpensive and user friendly optical fibre laser-inducedfluorescence (LIF) sensor based on fluorescence quenching by nanoparticles has beendeveloped to detect single-strand (ss-) DNA hybridization at femtomolar level. Thefluorescence of fluorophore-tagged ss-DNA increases by a factor of 80 when it binds to acomplimentary DNA, while the addition of single-base mismatch DNA had no effect on thefluorescence efficiency. We present theoretical and experimental results on dye fluorescencequenching induced by gold nanoparticles having different particle sizes. Fluorescencespectra clearly show that the quenching efficiency decreases with increasing size of thegold nanoparticles and increasing the distance between dye and nanoparticles.The mechanism of size- and distant-dependent fluorescence quenching has beendiscussed. Effects of various influential experimental parameters and configurationswere investigated in order to optimize and miniaturize the sensor performance.

Deposition of DNA-Functionalized Gold Nanospheres into Nanoporous Surfaces

We report the deposition of DNA-conjugated gold nanospheres into arrays of surface nanopores obtained from hexagonally ordered thin polystyrene-b-poly(methyl methacrylate) (PS-b-PMMA) diblock copolymer films on silicon. The deposition occurs spontaneously from aqueous solution and is driven by either electrostatic interactions or specific DNA hybridization events between the DNA nanospheres and the surface nanopores. To mitigate this spontaneous deposition, we have chemically modified the nanopores with either positively charged aminosilanes or oligonucleotide probe sequences. The deposition of DNA nanospheres into the surface nanopores was characterized by atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). We have observed preferential immobilization of individual DNA nanospheres within the nanopores, based on the size matching between the two entities. The inclusion density and selectivity of DNA nanosphere deposition into the surface nanopores was found to depend predominantly on the methods through which the nanoporous surfaces were prepared and chemically functionalized.

내열성 유전자 DgP23을 도입한 형질전환 오차드그라스의 생산

고온 내성 오차드그라스를 개발하기 위하여, 재조합 DgP23 유전자에 CaMV 35S 프로모터를 붙여 발현벡터를 제작, Agrobacterium 형질전환 방법으로 오차드그라스 형질전환체를 생산하였다. Genomic DNA를 분리하여 PCR 및 Southern blot 분석을 실시한 결과, PCR 분석에서 DgP23 유전자의 DNA band가 관찰되었고, Southern blot 분석에서도 X-ray film 상에 hybridization signal이 관찰되어, 오차드그라스 genome에 DgP23 유전자의 도입이 확인되었으며, wild type 및 empty vector control에서는 DNA band 및 hybridization signal이 관찰되지 않았다. 또한 RT-PCR 및 이들 산물의 Southern blot 분석 결과, DgP23 유전자의 정상적인 발현이 확인되었다. 형질전환 오차드그라스를 온실 및 포장에서 재배하며 생육특성을 조사한 결과, 비형질전환체와 비교하여 형태적 차이는 나타내지 않았다. 실험실 조건에서 고온내성을 조사한 결과, 고온내성이 확인되지 않았기 때문에 형질전환 종자를 생산하여 포장조건에서 고온내성을 검정할 계획이며, 재배시험에서는 내성이 강한 개체를 선발할 수 있을 것으로 예상된다. To develop transgenic orchardgrass (Dactylis glomerata L.) resistant to high temperature, a thermptolerance gene, DgP23, was introduced into orchardgrass using Agrobacterium - mediated transformation method. PCR and Southern blot analyses using genomic DNA showed specific DNA band on agarose gel and hybridization signal on X- ray film in transgenic orchardgrass harboring the recombinant DgP23 gene, but not in the wild type and empty vector control plants. RT-PCR and Southern blot analyses using total RNA also showed specific DNA band and hybridization signal. Transgenic orchardgrass did not showed ny morphological aberration both in the green house and field cultivation. Thermotolerance of transgenic plants was not detected in laboratory test. but may detected in field test.

Stability and hybridization-driven aggregation of silver nanoparticle–oligonucleotide conjugates

Gold nanoparticles are well known to form stable oligonucleotide conjugates, via thiol–metal interaction, which are capable of specific DNA-hybridization induced aggregation. Preparation of an analogous conjugate with Ag particles, except when coated with a layer of Au, has been reported to yield conjugates that are unstable in the hybridization environment. We report herein the hybridization-induced aggregation of such “bare” Ag-particle conjugates prepared via a similar facile procedure to Au with slight modification. We found that the pH during functionalization is the critical factor determining the success of preparing conjugates that remain stable throughout the functionalization process and during hybridization. We reasoned that this is a consequence of the pH-dependent charge of the oligonucleotide, and demonstrated that pH strongly affects the amount of oligonucleotides adsorbed on the particle surface, thereby imparting stability to the particles. This finding has the potential to be generalized to other metal particle–oligonucleotide systems with borderline stability, helping to expand the repertoire of visible-range plasmon signatures useful for diagnostic application.

Surface-Based Gold-Nanoparticle Sensor for Specific and Quantitative DNA Hybridization Detection

We present a sensitive and easily regenerated nano-optical sensor based on immobilization of avidin-coated colloidal gold particles on a biotin-modified planar lipid bilayer supported on the walls of a quartz cuvette. The so constructed sensing template, being specific for capturing of biotinylated biomacromolecules, is analyzed using optical spectroscopy combined with Mie theory calculations for quantification of the colorimetric changes induced by biorecognition events in the interfacial region of the particles. By further utilizing de Feiter's formalism, which correlates changes in effective refractive index and thickness with adsorbed mass, a good agreement between the Mie theory and experiments is demonstrated. Furthermore, the template is proven sensitive enough to follow the hybridization kinetics of 15-mer fully complementary DNA strands without the introduction of labels or secondary signal amplification.

RT-PCR-ELISA for detection of Apple stem grooving virus in apple trees

A method to detect Apple stem grooving virus (ASGV) based on reverse transcription polymerase chain reaction (RT-PCR) was developed using primers ASGV4F-ASGV4R targeting the viral replicase gene, followed by a sandwich hybridisation, in microtiter plates, for colorimetric detection of the PCR products. The RT-PCR was performed with the Titan™ RT-PCR system, using AMV and diluted crude extracts of apple (Malus domestica) leaf or bark for the first strand synthesis and a mixture of Taq and PWO DNA polymerase for the PCR step. The RT-PCR products is hybridised with both a biotin-labelled capture probe linked to a streptavidin-coated microtiter plate and a digoxigenin (DIG)-labelled detection probe. The complex was detected with an anti-DIG conjugate labelled with alkaline phosphatase. When purified ASGV was added to extracts of plant tissue, as little as 400 fg of the virus was detected with this method. The assay with ASGV4F-ASGV4R primers specifically detected the virus in ASGV-infected apple trees from different origins, whereas no signal was observed with amplification products obtained with primers targeting the coat protein region of the ASGV genome or with primers specific for Apple chlorotic leaf spot virus (ACLSV) and Apple stem pitting virus (ASPV). The technique combines the power of PCR to increase the number of copies of the targeted gene, the specificity of DNA hybridization, and the ease of colorimetric detection and sample handling in microplates.

Induction of premature chromosome condensation by a phosphatase inhibitor and a protein kinase in unstimulated human peripheral blood lymphocytes: a simple and rapid technique to study chromosome aberrations using specific whole-chromosome DNA hybridization probes for biological dosimetry

We developed a simple and rapid method to study chromosome aberrations involving specific chromosomes using unstimulated human peripheral blood lymphocytes (HPBL). Premature chromosome condensation (PCC) was induced by incubating unstimulated HPBL in the presence of okadaic acid (OA, a phosphatase inhibitor), adenosine triphosphate (ATP), and p34 cdc2/cyclin B kinase [an essential component of mitosis-promoting factor (MPF)], which eliminated the need for fusion with mitotic cells. OA concentration and duration of incubation for PCC induction was optimized using mitogen-stimulated HPBL; a final concentration of 0.75 μM incubated for 3 h was optimum, resulting in approximately 20% PCC yield. In unstimulated HPBL, PCC was induced by the addition of p34 cdc2/cyclin B kinase at concentrations as low as 5 units/ml to a cell culture medium containing OA. Increases in the concentration of p34 cdc2/cyclin B kinase from 5 to 50 units/ml resulted in a concentration-dependent increase in PCC yield (30% to 42%). We demonstrate that this technique of inducing PCC in unstimulated HPBL is suitable for studying radiation-induced aberrations involving a specific chromosome (chromosome 1) after 24 h repair using a whole-chromosome in situ hybridization probe and chromosome painting. Cells with aberrant chromosome number 1 are characterized with more than two chromosome spots. The frequency of cells with aberrant chromosome 1 increased with 60Co gamma-radiation doses in the region 0–7.5 Gy. The observed dose–effect relationship for the percentage of cells with aberrant chromosome 1 ( Y) was explained by using both a linear [ Y=(2.77±0.230) D+0.90±0.431, r 2=0.966] and a nonlinear power [ Y=(5.70±0.46) D (0.61±0.05), r 2=0.9901) model. This technique can be applied to biological dosimetry of radiation exposures involving uniform whole-body low linear energy transfer (LET) exposures.

Detection of DNA Hybridization Using the TISPR-1 Surface Plasmon Resonance Biosensor

Biotinylated oligonucleotide probes were immobilized to the gold sensor surface of the TISPR-1 miniature integrated surface plasmon resonance liquid sensor system for the purpose of detecting specific DNA hybridization. The immobilization of the oligonucleotide capture probes was carried out through streptavidin–biotin binding technology. The sensor detected the immobilization of unlabeled DNA through shifts in index of refraction as the molecules entered and remained selectively bound to the surface in the vicinity of the exponentially decaying surface plasmon resonance wave. The surface immobilization chemistry was proven to be stable for long periods of time, reproducible, and practical for detecting DNA hybridization with the TISPR-1. DNA hybridization was detected as a slow, positive, and small (when compared to protein–protein or antibody–antigen binding experiments) increase in the measured index of refraction under passive hybridization conditions by the TISPR-1 sensor. The DNA hybridization signal was significant (index of refraction change of 0.001) when large fragment PCR-amplified DNA products were hybridized to the oligonucleotide probes (S/N = 6–10). The DNA hybridization techniques were demonstrated using DNA sequences from the HIV genome which encode the Tat and Rev genes.

Localization of ribosomal RNA (rRNA), rRNA genes and silver staining nucleolar proteins in in vitro produced bovine embryos

In this paper the in vitro synthesis and application of non-radioactively labelled strand specific DNA probes is described. The probe is labelled by incorporation of nucleotides with the hapten digoxigenin into single-stranded DNA during a ‘run-off’ reaction catalyzed by Thermus aquaticus (Taq) DNA-polymerase. The ‘run-off’ reaction requires a linearized plasmid template and one primer binding site at a defined distance from the restriction site. Single-stranded DNA can be synthesized during repeated cycles of denaturation, annealing, and extension. The conditions for the incorporation of digoxigenin-11-dUTP (dig-11-dUTP) during polymerization were optimized to generate strand specific DNA hybridization probes up to a length of 5000 nt. The strand specificity is demonstrated by a dotblot, with in vitro-transcribed target RNA of c-sis. The sensitivity of the probe was tested in a Northern blot, and found to be identical to a probe radiolabelled by nick-translation (specific activity 6·5 × 108 cpm μg−1). The resolution of the signals and speed of development was even superior compared to the radiolabelled probe.

Model Systems for Developing Detection Methods for Foods Deriving from Genetic Engineering

Model systems for investigating the influence of the food matrix and depth of processing on detection limits for foods deriving from genetic engineering were established using the phytase-encoding gene ofEscherichia coliATCC 33965. For detection the polymerase chain reaction was applied directly to DNA extracted from foods based on cereals.E. colicells andE. coliDNA were added to musli, rolled oats, and rye flour in concentrations of 1010to 102CFU and 10 mg to 10 pg per gram, respectively. After soaking of the food samples in water or yoghurt, detection limits were determined to be 103to 104CFU and 10 to 100 pg by specific DNA hybridization. After sourdough fermentation of rye flour a hybridization signal was observed only with rather highE. coliconcentrations (>1010CFU), whereas detection was impossible in ready-baked breads.

Chromosome specific DNA hybridization in suspension for flow cytometric detection of chimerism in bone marrow transplantation and leukemia

Flow cytometry was used to measure the fluorescence intensity of nuclei that were subjected to fluorescent in situ hybridization in suspension with chromosome specific DNA probes. Paraformaldehyde-fixed nuclei were protein digested with trypsin and hybridized simultaneously with a biotin- and DIG labeled chromosome specific centromere probe. A number of probes were tested in the suspension hybridizations. The method yielded fluorescent hybridization signals that allow discrimination between Y chromosome positive and negative nuclei when analyzed by flow cytometry. The method is especially suited for analysis of bone marrow cells derived from patients who have received a sex-mismatched allogeneic bone marrow transplantation. Male leukemia cells with a trisomy for chromosome 8 were mixed with normal female cells and simultaneously hybridized in suspension with a DIG labeled probe specific for chromosome 8 and the biotin labeled Y chromosome probe. Y chromosome positive or negative nuclei were sorted onto microscope slides and subsequently classified as being leukemic or not by fluorescence microscopy, on the basis of the presence of a trisomy for chromosome 8. A 120-fold enrichment could be achieved when 300 Y positive nuclei were sorted from a mixture originally containing 0.5% leukemia cells. Given the specificity of the flow cytometry and FISH procedure, the combination of the two methods can reach a lower detection level of 1 per 250,000.

‘Run-off’ polymerization with digoxigenin labelled nucleotides creates highly sensitive and strand specific DNA hybridization probes: synthesis and application

In this paper the in vitro synthesis and application of non-radioactively labelled strand specific DNA probes is described. The probe is labelled by incorporation of nucleotides with the hapten digoxigenin into single-stranded DNA during a ‘run-off’ reaction catalyzed by Thermus aquaticus ( Taq) DNA-polymerase. The ‘run-off’ reaction requires a linearized plasmid template and one primer binding site at a defined distance from the restriction site. Single-stranded DNA can be synthesized during repeated cycles of denaturation, annealing, and extension. The conditions for the incorporation of digoxigenin-11-dUTP (dig-11-dUTP) during polymerization were optimized to generate strand specific DNA hybridization probes up to a length of 5000 nt. The strand specificity is demonstrated by a dotblot, with in vitro-transcribed target RNA of c-sis. The sensitivity of the probe was tested in a Northern blot, and found to be identical to a probe radiolabelled by nick-translation (specific activity 6·5 × 10 8 cpm μg −1). The resolution of the signals and speed of development was even superior compared to the radiolabelled probe.

Specific liquid DNA hybridization kinetics measured by fluorescence polarization

The kinetics of specific DNA hybrid formation were monitored directly in solution. Detection was based on fluorescence polarization measurements of labelled oligonucleotides at different stages during the hybridization. The effect of mismatched base pairs on the kinetics was measured. Significant differences could be observed in the kinetics when as few as three mismatches were introduced by the polymerase chain reaction technique in the target DNA sequences.

Development of a Colorimetric, Second Generation Nucleic Acid Hybridization Method For Detection of Salmonella in Foods and a Comparison With Conventional Culture Procedure

ABSTRACTA new, highly specific colorimetric DNA hybridization method (cDNAH) for detection of Salmonella in foods was developed. It detected 355/362 strains representing 206/213 serovars of Salmonella, and showed no cross‐reactivity with 32 different non‐Salmonella species, including a variety of Enterobacter and Citrobacter species. The cDNAH method was compared with the BAM/AOAC culture method in two studies: (1) with 20 different inoculated and uninoculated foods overall agreement between the two methods was 99%. (2) with naturally contaminated foods at two independent sites agreement with culture was 99.8% where 9/404 samples were positive for Salmonella.

A specific DNA hybridization probe for detection of Pasteurella piscicida

A specific DNA hybridization probe for detection of Pasteurella piscicida

Molecular studies of T-lymphocytes from cattle infected with bovine leukemia virus

Although bovine leukemia virus (BLV) is mainly associated with infections of B-lymphocytes, we have previously reported the statistically significant increase in the T-lymphocytes obtained from BLV-infected asymptomatic aleukemic (AL) cattle. In this report the presence of BLV provirus in the DNA of immunoaffinity purified T-lymphocytes from AL animals was assessed using a highly specific radiolabelled ( 32P) BLV-DNA provirus probe and solid phase DNA hybridization. The BLV provirus was found in the DNA of the peripheral blood mononuclear cells of all AL animals tested and three of the four purified T-lymphocyte preparations from these animals. The purified T-lymphocyte preparations used in this study contained less than 4% detectable B-lymphocytes. One animal had no detectable B-lymphocytes in the purified T-lymphocyte preparation and the DNA from these cells also gave positive hybridization results. The lymphocyte blastogenesis assay was then used as an indicator of the functional ability of lymphocytes from these BLV-infected AL cattle to respond to mitogenic stimuli. The responsiveness of lymphocytes from these animals to the mitogens concanavalin A (Con A), phytohemagglutinin (PHA), and pokeweed mitogen (PWM) was comparable to that of lymphocytes from BLV-negative animals when changes in 3H-thymidine uptake (c.p.m.) were used as measurement of mitogenic-induced blastogenesis. This indicated that infection of the T-lymphocytes by BLV does not appear to alter the overall response of the lymphocyte populations to mitogenic stimuli. High levels of spontaneous blastogenesis in the absence of mitogenic stimulation were observed for lymphocyte preparations of AL animals. The reason for this proliferation of lymphocytes is unclear; however, sera from these AL animals were found to contain a blastogenesis-augmenting factor(s) when added to lymphocytes from BLV-negative control animals in the presence of Con A, PHA and PWM.

Alpha-actin and cytochrome c mRNAs in atrophied adult rat skeletal muscle.

Specific complementary DNA (cDNA) hybridization probes were used to estimate the levels of alpha-actin and cytochrome c mRNAs and also 18S rRNA in three models of skeletal muscle atrophy. After 7 days of hindlimb suspension, or immobilization, or denervation, protein content decreased 26-32% in all muscles studied except suspended fast-twitch muscle, which lost only half as much protein. alpha-Actin mRNA content decreased 51-66% and cytochrome c mRNA content decreased 42-61% in slow- and fast-twitch muscles in all three models of atrophy. However, total RNA content did not show similar directional changes; RNA content decreased 27-44% in suspended and immobilized muscle but was unchanged in denervated fast-twitch muscle. The results were interpreted to suggest that loss of weight-bearing function of skeletal muscle is a major factor affecting the levels of alpha-actin and cytochrome c mRNAs during muscle atrophy.

Epstein-Barr Virus Associated B Cell Lymphoproliferative Disorders Following Bone Marrow Transplantation

Abstract B cell lymphoproliferative disorders (BLPD) developed in eight patients following bone marrow transplantation (BMT) for leukemia (five patients) or immunodeficiency (three patients). Recipients of T depleted marrow from a mismatched donor were at particularly high risk of this complication. Six of 25 (24%) recipients of mismatched T depleted bone marrow developed BLPD. In contrast, none of 47 matched T depleted transplants, one of ten (10%) who received non-depleted marrow from an unrelated donor, and only one of 424 matched non-depleted transplants were associated with BLPD. Epstein-Barr virus (EBV) specific serology and DNA hybridization studies demonstrating five to 50 copies of EBV genome/cell in involved tissues implicate this virus as an associated etiologic agent. Restriction fragment length polymorphism (RFLP) and cytogenetic analysis of involved tissue demonstrated donor origin (five of seven) or host origin (two of seven). Histologic appearance was similar to EBV-induced polymorphic B cell proliferations described following solid organ transplantation, or which occur de novo in primary immunodeficiency. Six of seven patients with adequate tissue available for study were found to have monoclonal proliferations by: in situ immunofluorescence (six of seven), and/or immunoglobulin gene rearrangement, (four of six). Cytogenetic analysis of involved tissues from four patients showed a normal karyotype, whereas two had multiple clonal chromosomal abnormalities. Seven patients died despite aggressive attempts at therapy with combinations of antiviral, immunologic, and chemotherapeutic agents.

Epstein-Barr virus associated B cell lymphoproliferative disorders following bone marrow transplantation

B cell lymphoproliferative disorders (BLPD) developed in eight patients following bone marrow transplantation (BMT) for leukemia (five patients) or immunodeficiency (three patients). Recipients of T depleted marrow from a mismatched donor were at particularly high risk of this complication. Six of 25 (24%) recipients of mismatched T depleted bone marrow developed BLPD. In contrast, none of 47 matched T depleted transplants, one of ten (10%) who received non-depleted marrow from an unrelated donor, and only one of 424 matched non-depleted transplants were associated with BLPD. Epstein-Barr virus (EBV) specific serology and DNA hybridization studies demonstrating five to 50 copies of EBV genome/cell in involved tissues implicate this virus as an associated etiologic agent. Restriction fragment length polymorphism (RFLP) and cytogenetic analysis of involved tissue demonstrated donor origin (five of seven) or host origin (two of seven). Histologic appearance was similar to EBV-induced polymorphic B cell proliferations described following solid organ transplantation, or which occur de novo in primary immunodeficiency. Six of seven patients with adequate tissue available for study were found to have monoclonal proliferations by: in situ immunofluorescence (six of seven), and/or immunoglobulin gene rearrangement, (four of six). Cytogenetic analysis of involved tissues from four patients showed a normal karyotype, whereas two had multiple clonal chromosomal abnormalities. Seven patients died despite aggressive attempts at therapy with combinations of antiviral, immunologic, and chemotherapeutic agents.

Selective Gene Amplification in Mammalian Cells after Exposure to 60 Co g Rays, 241 Am a Particles, or uv Light

Simian Virus 40 wild type (SV40)-transformed Chinese hamster embryo cells (Co631) contain about five viral copies integrated per cell genome. These SV40 sequences were used as endogenous indicator genes to study the response of mammalian cells to radiation at the gene level. An increase in copy number was detected by dispersed cell blotting and Southern analysis in combination with specific DNA hybridization. All types of radiation tested induce a 15- to 25-fold amplification of SV40 sequences without producing intact virus. The amplification is dose dependent and increases with time after irradiation: a maximum effect is observed at Day 3 after alpha particle or uv exposure and at Day 6 after gamma-ray exposure. A RBE of 6 can be calculated for alpha particles if amplification rates at Day 3 are compared. However, when the maximum effect is considered independent of time, no difference between different types of radiation is observed. Southern blots of genomic DNA show that not all integrated SV40 sequences are amplified upon radiation. Amplified sequences are found either in restriction fragments of relatively high molecular weight or in unit size fragments. SV40 amplification is selective in that the amplification of other genes, e.g., of alpha-actin, dhfr (dihydrofolate reductase), and of two oncogenes of the ras family (Kirsten ras and Harvey ras), was below detection level.