柯楠因诱导寡聚腺嘌呤核苷酸双螺旋结构增强赛博绿Ⅰ荧光及柯楠因的高灵敏和高选择荧光检测

柯楠因是原小檗碱类生物碱类似物.研究发现,在近生理酸度下柯楠因与寡聚腺嘌呤核苷酸(poly A16)形成双链结构,明显增强DNA嵌入染料赛博绿I(SG I)的荧光.在优化条件下,530 nm处荧光增强与60～600 nmol/L的柯楠因呈线性关系,检测限(3σ)为30 nmol/L.方法选择性好,同倍含量的小檗碱类似物不产生干扰.将所建立的方法用于柯楠因合成样的测定,回收率在92.5%～105%之间,相对标准偏差RSD小于3.1%(n=3).     

Interaction of cyanine dyes with nucleic acids: XXVI. Intercalation of the trimethine cyanine dye cyan 2 into double-stranded DNA: study by spectral luminescence methods

The interaction between double-stranded (ds) DNA and the cyanine dye Cyan 2 has been studied with spectral luminescence methods. Binding constant values have been determined by fluorescence titration and dye distribution in the two-phase system ethyl acetate-water (3.6 x 10(4) and 1.5 x 10(4) M(-1), respectively). Cyan 2 exhibits a small specificity for guanine-cytosine (GC) sequences in total DNA and synthetic polydeoxynucleotides poly(dA/dT) and poly(dGdC/dGdC). The DNA complexes with Cyan 2 are stable at high-ionic strength solution when NaCl is added. The dye molecule complexed with DNA is apparently shielded from the anionic quencher--iodide ion. The negative linear dichroism of the visible absorption band of aligned Cyan 2-DNA complexes indicates that the bound dye lies almost perpendicularly to the DNA helix axis. The linear dichroism of the absorption band at 260 nm suggests a considerable change in the DNA B-form. The results are consistent with an intercalative binding interaction between Cyan 2 and ds DNA.     

Monitoring viral DNA release with capillary electrophoresis

Viral DNA injection into host cells is one of the primary mechanisms of viral propagation. Drug development that targets viral propagation requires fast and sensitive methods for monitoring the release of viral DNA in vitro. Here we demonstrate the use of capillary electrophoresis (CE) for monitoring DNA release from virus particles. As a model for this study, we used T5 bacteriophages that infect the bacterium Escherichia coli K-12 by binding to the outer membrane FhuA receptor and then injecting DNA. DNA release from the T5 phages in vitro was induced by either elevated temperature or by interaction with the purified FhuA receptor. After DNA release, the viral samples were stained with the high affinity fluorescent dye YOYO-1, injected into the capillary and subjected to electrophoresis. YOYO-1-stained DNA generated a well-defined peak, allowing reliable detection of viral DNA from as few as 10(5) viral particles. The staining to track T5 phage DNA release exemplifies the great versatility that CE offers in studying viral systems. This CE-based method can be used to study molecular mechanisms of viral infections and to evaluate anti-viral drug candidates.     

Optimization of sequencing conditions using near-infrared lifetime identification methods in capillary gel electrophoresis

We have investigated the sample preparation and electrophoresis conditions necessary to prepare DNA sequencing samples appropriate for use with near-infrared (IR) fluorescent labels with dye identification accomplished via lifetime techniques. It was found that several sample preparation protocols required attention to maximize the fluorescence yields of the labeling dyes, such as thermal cycling conditions, choice of counter ion used for the ethanol precipitation step and also, dye-primer versus dye-terminator chemistries. In addition, several different sieving matrices were investigated for their effects on both the fluorescence properties of the labeling dyes and electrophoretic resolution. Extended times used for the high temperature denaturing of duplexed DNA fragments during cycle sequencing produced cleavage products, in which the covalently attached dye to the sequencing primer was released through attack by dithiothreitol (DTT). Even under optimized thermal cycling conditions, free dye was generated that masked readable data from the sequencing traces. Ethanol precipitation was necessary to remove this free dye with the proper choice of counter ion (sodium). The results using different sieving matrices indicated that linear polyacrylamides (LPAs) were appropriate for any fluorescence measurement, since they could readily be replaced between runs minimizing deleterious memory effects associated with cross-linked polyacrylamide gels. After investigation of several different sieving LPAs, the commercially available POP6 was found to be particularly attractive, since it produced good electrophoretic resolution, single exponential behavior for the near-IR dye series investigated herein, and also, discernible lifetime differences within the dye set. Finally, dye-terminator chemistry was also found to minimize bleeding in the gel matrix produced by large amounts of unextended dye-primer within the gel lane.     

Drawing and dynamic-mechanical behavior of syndiotactic polystyrene an introduction

The drawing behavior of syndiotactic polystyrene was analyzed at different temperatures. Amorphous films were used and, depending on drawing temperature, strain-induced or thermal-induced cold crystallization was observed. This phenomenon, when present, greatly affects the drawing behavior. The dynamic-mechanical behavior of drawn samples was analyzed, and the obtained results indicate that the glass transition is affected by drawing, and that the effect depends drastically on the drawing temperature. Particularly interesting is the dynamic behavior at high temperature where the elastic modulus is weakly affected by temperature also near the melting point.     

The pH stimulated reversible loading and release of a cationic dye in a layer-by-layer assembled DNA/PAH film

Through the layer-by-layer (LbL) deposition method, DNA was assembled into an ultrathin film with a cationic poly(allylamine hydrochloride) (PAH). The loading and release of a typical cationic dye, 5, 10, 15, 20-tetrakis(4-N-methylpyridyl)porphine-tetra-(p-toluenesulfonate) (TMPyP), in the DNA/PAH films were investigated. It has been found that the LbL-assembled DNA/PAH film was very stable in both acidic and alkaline solutions. Stimulated by the pH change of the dye solution, the dye can be easily loaded into or released from the DNA/PAH film. In an alkaline solution, the dye could be rapidly loaded into the DNA/PAH film at room temperature, while in an acidic solution, the dye could be rapidly released. The mechanism of such pH-stimulated loading and release in the DNA/PAH film was discussed. It was further observed that the loading and release of the dye in the DNA/PAH film was reversible upon pH change and the process could be repeated many times.     

Cetyltrimethylammonium bromide sensitized resonance light-scattering of nucleic acid--Pyronine B and its analytical application

This is the first report on the determination of nucleic acids with Pyronine B (PB) sensitized by cetyltrimethylammonium bromide (CTMAB) with resonance light-scattering (RLS) technique. Under the experimental conditions (1 x 10(-5) mol l(-1) PB, 1 x 10(-5) mol l(-1) CTMAB, pH 7.4, at room temperature, ionic strength 0.02 mol l(-1) NaCl), the interaction of PB with DNA sensitized by CTMAB results in enhanced RLS signals at 328 and 377 nm in the enhanced regions. It was found that the enhanced RLS intensity at 328 nm was proportional to the concentration of DNA in the suitable ranges. The linear range of this assay is 0.0-1.2 microg ml(-1) for calf thymus, 0.0-0.8 microg ml(-1) for fish sperm DNA (fsDNA), and 0.04-1.4 microg ml(-1) for yeast RNA, respectively. The detection limits (3 sigma) are 6.1 ng ml(-1) for calf thymus DNA (ctDNA), 11.2 ng ml(-1) for fish sperm DNA, and 8.6 ng ml(-1) for yeast RNA, respectively. Six synthetic samples were determined satisfactorily. This method is simple, rapid and the dye is inexpensive and stable.     

Determination of nucleic acids by near-infrared fluorescence quenching of hydrophobic thiacyanine dye in the presence of Triton X-100.

A near-infrared (near-IR) fluorescence quenching method was developed for the determination of nucleic acids in aqueous solution by using a cationic heptamethylene thiacyanine as a probe. The near-IR cationic cyanine showed maximum excitation and emission wavelengths at 800 and 825 nm, respectively, in the presence of Triton X-100; the fluorescence of the cyanine could be greatly quenched by DNA. The calibration graphs were linear over the range of 10-400 ng/mL for CT (calf thymus) DNA and over the range 5-400 ng/mL for FS (fish sperm) DNA under optimal conditions. The corresponding detection limits were 5.2 ng/mL for CT DNA and 2.5 ng/mL for FS DNA. The relative standard deviation (n = 8) was 3.1% for 75 ng/mL CT DNA and 2.2% for 75 ng/mL FS DNA, respectively. Preliminary research showed that the fluorescence quenching might be ascribed to the formation of dye aggregate facilitated by DNA.     

Plastic microchip electrophoresis for genetic screening: the analysis of polymerase chain reactions products of fragile X (CGG)n alleles

Clinical screening of abnormal chromosomes associated with fragile X syndrome (FXS) demands a high-throughput method including DNA sizing and detection of the amplified products. This study is to explore the use of polymer microchip electrophoresis for the analysis of polymerase chain reaction (PCR) products of fragile X (CGG)n alleles to facilitate a fast exclusion test of FXS. The sequences flanking the CGG-repeat of FMR1 gene was amplified by betaine-PCR and the amplified products were desalted and then analyzed by microchips which were fabricated on poly(methyl methacrylate) (PMMA) substrate. The PCR bands with more than six CGG-repeats in difference could be clearly distinguished in less than 3 min by microchip electrophoresis with a separation length of 6 cm. It was found that the signal was greatly enhanced with the use of both covalent (Cy5) and intercalating dye (TORRO-3), which has never been demonstrated before. We tested the method by reanalysis of twelve samples from males and six samples from females. For female samples with less than six repeat differences, Southern blotting method was performed to confirm or exclude the findings from microchips. It was found that the test results from all male and female samples show a 100% correlation between the microchip electrophoresis and the existing methods.     

分析测试新成果(230~237)2种常见燃油添加剂对汽油燃烧烟尘的影响

采用气相色谱-质谱(GC-MS)联用技术,根据汽油原样中的特征成分,对未加入和分别加入汽油燃油精、海龙燃油宝的汽油燃烧烟尘进行对比分析.结果表明,汽油燃油精的加入会使汽油燃烧烟尘中各类特征成分的百分含量有所变化,但对谱图和特征成分的影响较小;而海龙燃油宝的加入对汽油燃烧烟尘的谱图、特征成分及其个数、各类特征成分的百分含量均产生较大影响.结果为火灾物证鉴定提供一定的参考依据.     

Increased intensities of YOYO-1-labeled DNA oligomers near silver particles

DNA detection is usually performed using fluorescence probes. Using a DNA oligomer stained with the widely used dye 1,1'-[1,3-propanediylbis[(dimethylimino)-3,1-propanediyl]]bis[4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]]-quinolinum tetraiodide (YOYO-1), we show that a substrate containing silver particles can lead to a greater than 10-fold increase in the fluorescence intensity. Proximity to silver particles also increases the photostability of YOYO-1-DNA. These results suggest that substrates or gels containing silver particles may be used for increased sensitivity in DNA detection.     

Determination of Violet Covasol as a cosmetic dye in water samples by a CPE-Scanometry method

The trace amounts of Violet Covasol as a cosmetic dye was determined by an efficient cloud point extraction-Scanometry(CPE-Scanometry) method. This method has many advantages such as novelty,facility, high speed, sensitivity, low cost and safety. The method is based on the CPE of an analyte from an aqueous solution, diluting the extracted surfactant-rich phase with ethanol, transferring to Plexiglas~cell and scanning of the cells containing the analyte solution with a scanner and measuring the RGB parameters with software written in visual basic(VB 6) media. Parameters impacting the extraction efficiency such as p H of the system, the concentration of surfactant, equilibration temperature and time were optimized. Detection limit(DL), relative standard deviation(RSD) and linear range for the proposed method are 0.026, 0.71 and 0.16–6.6 μg m L ~(-1)respectively. The method was successfully applied for the determination of Violet Covasol dye in several water samples, including a water sample containing the dye as a tracer(to investigate subsurface water movement).     

A preliminary evaluation of serum proteins by capillary electrophoresis

We evaluated the 5-band Serum Proteins by Capillary Electrophoresis kit (Bio-Rad Laboratories, Hercules, CA, U.S.A.) on the BioFocus 2000 CE (Bio-Rad) against conventional agarose gel electrophoresis (AGE) (Helena Laboratories, Beaumont, TX, U.S.A.). Serum from 60 patients was initially screened by AGE and divided into three groups: 1) normal electrophoretic pattern (n = 36, mean total protein 67.7 g/L), 2) monoclonal/oligoclonal gammopathy (n = 14, mean total protein 78.8 g/L), and 3) polyclonal gammopathy (n = 10, mean total protein 77.4 g/L). These samples were concurrently analyzed on the BioFocus 2000. Intraassay and interassay CVs for the five fractions for a normal sample were 0.17-1.44% and 0.42-9.11%, respectively, and 0.21-3.37% and 0.29-3.61%, respectively, for a sample with monoclonal gammopathy. Correlation coefficients for albumin and the albumin/globulin (A/G) ratio were 0.8891 (albumin range 17-93 g/L) and 0.8276 (A/G ratio range 0.39-7.81), respectively. The A/G ratio alone could not discriminate between the three groups. Capillary zone electrophoresis (CZE) correctly identified 33 of 36 samples in the normal group, and 22 of 24 samples in the other two groups, giving a clinical sensitivity and specificity of 91.7%. Our preliminary evaluation shows that protein separation by CZE is a simple, rapid, and automated alternative to conventional AGE.     

分散染料染色法制备的彩色聚(苯乙烯-丙烯酸)纳米球

在低于聚合物纳米球玻璃化转变温度(110.69 ℃)的条件下,用两种分散染料对聚(苯乙烯-丙烯酸)[P(St-co-AA)]纳米球染色,研究了染色温度(75~95 ℃)和染料用量(1%~5%)对纳米球上染料吸附量的影响。 结果表明,染色温度越高,分散染料用量越大,所得到聚合物纳米球的颜色越深越鲜艳。 分子结构中氨基和羟基数量多的分散蓝2BLN在纳米球上的吸附量低于相同染料用量时分散红FB的吸附量。 经过染色后纳米球的表面变得很粗糙,粒径增加23 nm。 用彩色纳米球对经过阳离子化处理的棉织物进行染色,利用很少量的彩色纳米球,就可以使织物获得较深且鲜艳的颜色。     

Determination of Genetically Modified Soybean by Multiplex PCR and CGE with LIF Detection

A method for rapid screening, identification and detection of genetically modified soybean by multiplex polymerase chain reaction (PCR) and capillary gel electrophoresis with laser-induced fluorescence (CGE–LIF) was developed and applied to actual food samples. A triplex PCR procedure was used to amplify the parts of nopaline synthase (NOS) terminator, and the junction between cauliflower mosaic virus 35S promoter and chloroplast transit peptide CTP4 trait gene, as well as the lectin gene to allow the screening and identification of specific transgenic soybean line (glyphosate-tolerant soybean). The multiplex PCR parameters and conditions of capillary gel electrophoresis were optimized. The amplified DNA fragments were analyzed by CGE–LIF. The amplified PCR products were analyzed by CGE–LIF within about 20 min. The method developed is highly sensitive and allows the detection of a percentage of genetically modified soybean as low as 0.025%. The percentage is low enough to fulfill the requirement of the EU Regulation for transgenic food labeling of 1.0%. The sequences of the multiple PCR products were identical with those published in Genbank. The proposed method has been used in identification and detection of genetically modified soybean in various food samples. Compared with agarose gel electrophoresis (AGE), the proposed method is more rapid, accurate and requires a smaller amount of samples. Thus an efficient alternative method is provided for monitoring genetically modified soybean in order to meet the increasing demand of implementation of the genetically modified food labeling policy.     

Nuclease P1 digestion/high-performance liquid chromatography, a practical method for DNA quantitation

We have developed a practical method for quantifying DNA. The method is practical in two ways. First, a single enzyme is used to digest the DNA to nucleotides that are then quantified by HPLC under ordinary conditions. Second, the method quantifies DNA even when it is impure. In our method, "nuclease P1/HPLC," the DNA is hydrolyzed by nuclease P1 and the resulting 2'-deoxynucleoside 5'-monophosphates are quantified by HPLC with UV detection. This method was applied to several kinds of genomic DNA in terms of origin and method by which it had been purified. Calf thymus DNA (purified by salt precipitation by the supplier), pig liver DNA (purified by phenolic extraction or by anion-exchange chromatography using a Genomic Tip from Qiagen) and mouse skin DNA (similarly purified) were tested. In some cases a given sample was purified by two of these methods. The values for the amount of DNA by our method were compared with those by three other methods: acid hydrolysis/HPLC (selected as a reference procedure), UV absorbance, and dye binding. Agreement for all DNA samples between the values by our method versus those provided by acid hydrolysis/HPLC was within 10% for amounts of DNA in the 19-54 microg range. In contrast, UV absorbance and the dye-binding assay gave differences up to 30-40% relative to the consistent values furnished by acid hydrolysis and our method. Overall, normalizing the concentrations of the DNA (thymus, liver, skin) by acid hydrolysis/HPLC in 10 samples to values of 1.0 gave the following, relative values and standard deviations: 1.01+/-.07 (nuclease P1/HPLC), 0.8+/-0.17 (dye binding), and 1.1+/-0.1 (UV). Since one cannot assume that any sample of DNA is pure, and determining purity of DNA is difficult, then nuclease P1/HPLC or acid hydrolysis/HPLC is recommended rather than the UV absorbance or dye binding for quantifying DNA whenever an accurate value is important.     

Noncontact temperature measurement in microliter-sized volumes using fluorescent-labeled DNA oligomers

A new method of noncontact temperature measurement in microliter-sized volumes is demonstrated, based on the temperature sensitivity of the fluorescence lifetime of rhodamine-G when it is attached to a DNA oligomer. As temperature changes, the spacing between the fluorescent dye and a designed sequence of DNA bases is modulated by conformation changes of the DNA chain, and as a result the ability of dye molecules to fluoresce is also modulated according to differential quenching by bases on the DNA. In the system that we studied, the temperature sensitivity of the fluorescence lifetime was 36-42 ps/ degrees C depending on specific solution conditions. Although this strategy of temperature measurement is demonstrated using a specific sequence of DNA, it can also be generalized to a dye attached to any other intrinsic quencher of fluorescence whose conformation changes with temperature.     

FLUORESCENCE OF COMPLEXES OF QUINACRINE MUSTARD WITH DNA. I. INFLUENCE OF THE DNA BASE COMPOSITION ON THE DECAY TIME IN BACTERIA

The fluorescence of several bacterial DNAs stained with quinacrine mustard have been investigated using a laser microfluorometer with a spatial resolution of - 0.3 μm and a temporal resolution of ?0.3 ns connected to a digital signal averager. Experiments performed on Micrococcus lysodeikticus samples show that both cytological preparations and the corresponding purified DNAs give coincident fluorescence curves, thus indicating that the fluorescence observed in the former case is to be attributed to the bacterial DNA only. Experiments thereafter performed on smears of several bacteria with known AT percentages show that each fluorescence decay curve, after a fast transient, can be fitted by an exponential decay law with a single time constant. This time constant has been found to depend linearly on the square of the AT percentage. We explain this result on the basis of an energy transfer mechanism between dye molecules intercalating AT:AT sequences (donors) and dye molecules bound to either GC:GC or GC:AT sequences (acceptors). The agreement with the experimental data requires that all the bacteria considered present a common value for both the number of base pairs contained in a Förster sphere and for the maximum saturation of the strong binding process.     

Fluorescence detection of single-nucleotide polymorphisms with two simple and low cost methods: a double-DNA-probe method and a bulge form method

Two 10-mer DNA probes, or one 20-mer DNA probe, respectively, hybridize with a 21-mer target DNA to form a vacancy or bulge opposite the target nucleotide. The former double-DNA-probe method and the latter bulge form method are applicable to the detection of single-nucleotide polymorphisms (SNPs). A small fluorescent dye enters into the vacancy or bulge and binds with a target nucleotide via a hydrogen bonding interaction, which causes fluorescence quenching. The interaction between fluorescent dye and the target nucleotide is confirmed by measuring the melting temperature and fluorescence spectra. The fluorescent dye, ADMND (2-amino-5,7-dimethyl-1,8-naphthyridine), is found to selectively bind with C over A or G. The methods proposed here are economic, convenient, and effective for the fluorescence detection of SNPs. Finally, the double-DNA-probe method and bulge form method are successfully applied to the detection of C/G and C/A mutations in the estrogen receptor 2 gene and progesterone receptor gene using ADMND.     

Suppression Of Ages Formation In Spontaneous Diabetic OLETF Rat By Organic Germanium Compound [Poly-trans-(2-carboxyethyl) Germasesquioxane] (Ge-132)

The model rat for the type 2 diabetes mellitus (NIDDM) in human were observed for 72 weeks after birth, administering an organic germanium compound [Bis(2-Carboxyethyl) germasesquioxane, Ge-132] perorally 100 mg/kg/day since 24 weeks old. Clinical examinations were followed throughout the observation period. Blood and urinary glucose in positive control OLETF rats tended to be higher than those treated with Ge-132. At the end of the 72nd week, animals were sacriificed to examine the pathological changes, specifically in pancreas, kidney and brain. Anti-AGE antibody stained proximal and distal tubles and basement membrane of glomerules in kidney, and accumulated AGE masses in cortex, hippocampus and cerebellum of OLETF rat's brain. Amyloid stains by basic congo red on kidney and brain revealed that the deposits of amyloid in kidney mesangium and in cortex, hippocampus and cerebellum were observable in OLETF rats. Ge-132 suppressed the deposition of amyloid tangles in kidneys and brains. Anti rat complement C'3 antibody reacted with AGE and amyloid tangles that were sensitive to anti-AGE antibody. AGE generated in vitro by incubating human serum, human gammaglobulin (HGG), or bovine serum albumin (BSA) with glucose activated complements, showing the consumption of complements in the hemolysis of hemolysin-coated sheep red blood cells. A novel device Quantum Resonance Spectrometer (QRS) could read the subtle bio-magnetism memorized in serum samples, demonstrating quantitative values reflecting the patho-physiology of OLETF rats.