Quantitative nonisotopic nitrocellulose filter binding assays: bacterial manganese superoxide dismutase-DNA interactions

Nitrocellulose filter binding assays (NCFBAs) have been used for many years to qualitatively and quantitatively determine protein-nucleic acid affinities. While this technique can be robust thermodynamically and fairly simple to perform, the requirement of radiolabeled nucleic acids (typically (32)P) has several major drawbacks. Some disadvantages are the short half-life of (32)P, the inherent safety concerns, and the cost of working with radioisotopes. Another drawback is that over time the beta emissions cause fragmentation of the nucleic acids. We have modified standard NCFBAs by developing a quantitative nonisotopic chemiluminescent method using biotin-labeled DNA and a dual-filter format. The biotin tag is detected on both nitrocellulose and positively charged nylon membranes by conventional streptavidin-conjugated alkaline phosphatase recognition systems. Quantitation of the photon emissions is simplified by use of a cooled charge-coupled device camera, although exposure to X-ray film and quantitation by densitometry could also be employed. The binding affinity of bacterial manganese superoxide dismutase to nonsequence-specific DNA has been quantitated previously by standard NCFBAs. We have replicated parts of a published binding study using identical solution conditions and the nonisotopic method that we have developed. We provide quantitative agreement between the isotopic and the nonisotopic methods.     

Microwave irradiation-stimulated in situ hybridization procedure with biotinylated DNA probe.

A microwave-stimulated in situ hybridization technique using biotin-labeled DNA probe is described. Both hybridization reaction and the detection of the biotin label (with a alkaline phosphatase or immunofluorescence method) has been performed in the microwave oven. All procedures are completed within one hour. The described method was applied for identification of nucleic acid sequences of human immunodeficiency virus in human cell lines. The resolution and the intensity of the signal are as good as from a standard technique with overnight incubation of the probe. Because of the simplicity and speed of the technique, this procedure can be used in a number of other applications.     

Detection of microarray-hybridized oligonucleotides with magnetic beads

The efficiency of hybridization analysis with oligonucleotide microarrays depends heavily on the method of detection. Conventional methods based on labeling nucleic acids with fluorescent, chemiluminescent, enzyme, or radioactive reporters suffer from a number of serious drawbacks which demand development of new detection techniques. Here, we report two new approaches for detection of hybridization with oligonucleotide microarrays employing magnetic beads as active labels. In the first method streptavidin-coated magnetic beads are used to discover biotin-labeled DNA molecules hybridized with arrayed oligonucleotide probes. In the second method biotin-labeled DNA molecules are bound first to the surface of magnetic beads and then hybridized with arrayed complementary strands on bead-array contacts. Using a simple low-power microscope with a dark-field illumination and a pair of complementary primers as a model hybridization system we evaluated sensitivity, speed, and cost of the new detection method and compared its performance with the detection techniques employing enzyme and fluorescent labels. It was shown that the detection of microarray-hybridized DNA with magnetic beads combines low cost with high speed and enhanced assay sensitivity, opening a new way to routine hybridization assays which do not require precise measurements of DNA concentration.     

毛细管内均相辣根过氧化物酶（HRP）与核酸杂交偶联HRP催化化学发光比较研究

目的：了解毛细管内核酸偶联和游离辣根过氧化物酶（HRP）催化化学发光差异。方法：不同浓度HRP和经核酸杂交固定于毛细管内壁HRP催化化学发光反应。结果：①游离HRP催化化学发光线性检测范围窄（2.7×10-5-1.3×10-6mg/ml,R2〉0.96）,下限为1.0×10-7mg/ml;②2.0×1014-2.0×106copies/ml的5μl单链DNA杂交后,1.0-10min时DNA浓度M对数（lgM）与化学发光I值线性相关（R2〉0.99）,且大于阴性I值平均数＋3倍标准偏差（s.d）;③5.0×1011-5.0×106copies/ml的5μl PCR产物杂交后,10min内PCR产物对数lgM与I值线性相关（R2〉0.97）,且大于阴性I值平均数＋3倍标准偏差（s.d）。4.0-7.0min内lgM与I值的R2〉0.99,3次平行检测标准偏差〈5.0%。结论：毛细管内核酸杂交的HRP催化化学发光检测线性范围宽、灵敏度高、底物用量少,有望用于临床核酸分子杂交检测。     

Rapid detection of human papilloma virus using a novel leaky surface acoustic wave peptide nucleic acid biosensor

A novel leaky surface acoustic wave (LSAW) bis-peptide nucleic acid (bis-PNA) biosensor with double two-port resonators has been constructed successfully for the quantitative detection of human papilloma virus (HPV). The bis-PNA probe can directly detect HPV genomic DNA without polymerase chain reaction (PCR) amplification, and it can bind to the target DNA sequences more effectively and specifically than a DNA probe. When the concentrations varied from 1 pg/L to 1000 μg/L, with 100 μg/L being the optimal, a typical linearity was found between the quantity of target and the phase shifts. The detection limit was 1.21 pg/L and the clinical specificity was 97.22% of that of real-time PCR. The bis-PNA probe was able to distinguish sequences that differ only in one base. Both the intraassay and interassay coefficients of variance (CVs) were <10%, and the biosensor can be regenerated for ten times without appreciable loss of activity. Therefore, this technical platform of LSAW biosensor can be applied to clinical samples for direct HPV detection.     

Measurement of telomere length by the Southern blot analysis of terminal restriction fragment lengths

In this protocol we describe a method to obtain telomere length parameters using Southern blots of terminal restriction fragments (TRFs). We use this approach primarily for epidemiological studies that examine leukocyte telomere length. However, the method can be adapted for telomere length measurements in other cells whose telomere lengths are within its detection boundaries. After extraction, DNA is inspected for integrity, digested, resolved by gel electrophoresis, transferred to a membrane, hybridized with labeled probes and exposed to X-ray film using chemiluminescence. Although precise and highly accurate, the method requires a considerable amount of DNA (3 μg per sample) and it measures both the canonical and noncanonical components of telomeres. The method also provides parameters of telomere length distribution in each DNA sample, which are useful in answering questions beyond those focusing on the mean length of telomeres in a given sample. A skilled technician can measure TRF length in ～130 samples per week.     

PCR-Free Detection of Genetically Modified Organisms Using Magnetic Capture Technology and Fluorescence Cross-Correlation Spectroscopy

The safety of genetically modified organisms (GMOs) has attracted much attention recently. Polymerase chain reaction (PCR) amplification is a common method used in the identification of GMOs. However, a major disadvantage of PCR is the potential amplification of non-target DNA, causing false-positive identification. Thus, there remains a need for a simple, reliable and ultrasensitive method to identify and quantify GMO in crops. This report is to introduce a magnetic bead-based PCR-free method for rapid detection of GMOs using dual-color fluorescence cross-correlation spectroscopy (FCCS). The cauliflower mosaic virus 35S (CaMV35S) promoter commonly used in transgenic products was targeted. CaMV35S target was captured by a biotin-labeled nucleic acid probe and then purified using streptavidin-coated magnetic beads through biotin-streptavidin linkage. The purified target DNA fragment was hybridized with two nucleic acid probes labeled respectively by Rhodamine Green and Cy5 dyes. Finally, FCCS was used to detect and quantify the target DNA fragment through simultaneously detecting the fluorescence emissions from the two dyes. In our study, GMOs in genetically engineered soybeans and tomatoes were detected, using the magnetic bead-based PCR-free FCCS method. A detection limit of 50 pM GMOs target was achieved and PCR-free detection of GMOs from 5 µg genomic DNA with magnetic capture technology was accomplished. Also, the accuracy of GMO determination by the FCCS method is verified by spectrophotometry at 260 nm using PCR amplified target DNA fragment from GM tomato. The new method is rapid and effective as demonstrated in our experiments and can be easily extended to high-throughput and automatic screening format. We believe that the new magnetic bead-assisted FCCS detection technique will be a useful tool for PCR-free GMOs identification and other specific nucleic acids.     

自制显示系统检测免疫印迹中的目的蛋白

目前蛋白免疫印迹显示系统有显色法和化学发光法,因化学发光法具有灵敏、快速、准确等特点而倍受科研工作者的青睐,但一般商品化的化学发光试剂盒价格颇为昂贵,为此,在参考文献的基础上作了一些改进,将Tris Cl的pH值从7.5提高到11.0,同时提高了对碘苯酚的浓度（从1mmol/L提高到2mmol/L）,得到了自制的化学发光试剂。首先用其检测了大肠杆菌中表达的2种不同分子量的蛋白（gam,13kDa;bet,30kDa）,发现当蛋白上样量为2~20ng时,各蛋白条带均清晰显示。随后的斑点免疫印迹实验表明：自制及进口化学发光试剂在检测酶标二抗效价方面有着同样高的敏感性。     

Competition dialysis: a method for the study of structural selective nucleic acid binding

Competition dialysis is a powerful new tool for the discovery of ligands that bind to nucleic acids with structural- or sequence-selectivity. The method is based on firm thermodynamic principles and is simple to implement. In the competition dialysis experiment, an array of nucleic acid structures and sequences is dialyzed against a common test ligand solution. After equilibration, the amount of ligand bound to each structure or sequence is determined by absorbance or fluorescence measurements. Since all structures and sequences are in equilibrium with the same free ligand concentration, the amount bound is directly proportional to the ligand binding affinity. Competition dialysis thus provides a direct and quantitative measure of selectivity, and unambiguously identifies which of the samples within the array are preferred by a particular ligand. We describe here the third generation implementation of the method, in which competition dialysis was adapted for use in a 96-well plate format. In this format, we have been able to greatly expand the array of nucleic acid structures studied, and now can routinely study the interactions of a ligand of interest with 46 different structures and sequences.     

检测HSA—GLP-1融合蛋白双抗体夹心ELISA方法的建立

目的：为检测血清中HSA-GLP-1融合蛋白整体分子的浓度,建立一种特异、灵敏的定量检测食蟹猴体内HSA-GLP-1融合蛋白浓度的双抗体夹心ELISA的方法。方法：采用双抗体夹心ELISA方法,以GLP-1单克隆抗体为包被抗体、HSA-GLP-1融合蛋白为夹心抗原、anti-HSA-Biotin为检测抗体,用Streptavidin-HRP进行免疫放大,TMB显色。结果：建立了检测HSA-GLP-1融合蛋白的ELISA方法,其线性范围为15.6~1000 ng/mL,最低检测限为15.6 ng/mL,与GLP-1、HSA、IL2-HSA均无交叉反应,板内和板间精密度均小于15%,准确度为±15%,冻融稳定性和稀释稳定性良好。结论：建立的HSA-GLP-1蛋白检测方法符合新生物制品临床前药代动力学研究指导原则要求,可用于HSA-GLP-1融合蛋白在临床前药代动力学试验的定量检测。     

Nucleic acid-based fluorescent probes in microbial ecology: application of flow cytometry

Microorganisms in natural environments have often been treated as 'black box' systems. Researchers have measured the inputs and outputs of the box, and have made bulk measurements on cell behaviour. However, unravelling the details of the diversity and interactions that exist within these microbial populations has proven exceptionally difficult. The information gained from the black box approach has been invaluable, and has allowed models of global foodwebs to be generated and tested. However, there is still little information about the interactions of individual microbial cells within natural populations. Such studies are essential to fully understand the integrated functioning of ecosystems. To achieve this goal, researchers need to be able to identify individual cells within a population, enumerate them, estimate both viability and activity, and monitor changes in response to relevant parameters. Due to the diversity, heterogeneity and numbers of cells that make up these populations, these measurements require automation and speed. At present, the use of flow cytometry in conjunction with nucleic acid probes provides an excellent method with which to pursue such studies.     

The Jalview Java alignment editor

Multiple sequence alignment remains a crucial method for understanding the function of groups of related nucleic acid and protein sequences. However, it is known that automatic multiple sequence alignments can often be improved by manual editing. Therefore, tools are needed to view and edit multiple sequence alignments. Due to growth in the sequence databases, multiple sequence alignments can often be large and difficult to view efficiently. The Jalview Java alignment editor is presented here, which enables fast viewing and editing of large multiple sequence alignments.     

A high throughput method to investigate oligodeoxyribonucleotide hybridization kinetics and thermodynamics.

We describe a high throughput microtiter-based assay to measure binding of oligodeoxyribonucleotides to nucleic acid targets. The assay utilizes oligodeoxyribonucleotide probes labeled with a highly chemiluminescent acridinium ester (AE). Reaction of AE with sodium sulfite renders it non-chemiluminescent. When an AE-labeled probe hybridizes to a target nucleic acid AE is protected from reaction with sodium sulfite and thus remains chemiluminescent. In contrast, unhybridized probe readily reacts with sodium sulfite and is rendered non-chemiluminescent. Hybridization of an AE-labeled probe to a target nucleic acid can therefore be detected without physical separation of unhybridized probe by treatment of the hybridization reaction with sodium sulfite and measurement of the remaining chemiluminescence. Using this method we measured hybridization rate constants and thermodynamic affinities of oligodeoxyribonucleotide probes binding to simple synthetic targets as well as large complex biological targets. The kinetic and thermodynamic parameters were measured with a high degree of accuracy and were in excellent agreement with values measured by other established techniques.     

Determination of protein-bound phosphate by continuous flow analysis: Automated procedure for the determination of alkali-labile phosphate

A method is described for the specific, quantitative determination of protein-bound phosphorus by a continuous flow procedure using a Technicon AutoAnalyzer. It is based on the exceptional alkali lability of serine phosphate linkages to β-elimination when the serine residues are present in a polypeptide chain. The results are reproducible within about 3, 5, or 10%, respectively, when the analytical sample contains about 100, 10, or 3 nmol of protein-bound P. The presence of less than 1 nmol protein-bound P can be detected. The method tolerates wide variations of the pH and ionic composition of the sample, making it suitable for the automatic, serial analysis of chromatographic effluent fractions. Low-molecular-weight phosphomonoesters, ribonucleic acid (phosphodiester), and nucleotide phosphates (pyrophosphate) do not react measurably. Carboxyphenyl phosphate is partially hydrolyzed (10–15%). In contrast, the release of P from various phosphoproteins is quantitative.     

Hydroxyl radical in living systems and its separation methods

It has recently been shown that hydroxyl radicals are generated under physiological and pathological conditions and that they seem to be closely linked to various models of pathology putatively implying oxidative stress. It is now recognized that the hydroxyl radical is well-regulated to help maintain homeostasis on the cellular level in normal, healthy tissues. Conversely, it is also known that virtually every disease state involves free radicals, particularly the most reactive hydroxyl radical. However, when hydroxyl radicals are generated in excess or the cellular antioxidant defense is deficient, they can stimulate free radical chain reactions by interacting with proteins, lipids, and nucleic acids causing cellular damage and even diseases. Therefore, a confident analytical approach is needed to ascertain the importance of hydroxyl radicals in biological systems. In this paper, we provide information on hydroxyl radical trapping and detection methods, including liquid chromatography with electrochemical detection and mass spectrometry, gas chromatography with mass spectrometry, capillary electrophoresis, electron spin resonance and chemiluminescence. In addition, the relationships between diseases and the hydroxyl radical in living systems, as well as novel separation methods for the hydroxyl radical are discussed in this paper.     

Quantitative aspects of cytochemical methods for acetylcholinesterase studied with a cytochemical model system

A model system of polyacrylamide films containing the Triton extract of rat brain homogenate was applied to investigate quantitatively some aspects of three methods for the cytochemical demonstration of acetylcholinesterase activity (Lewis 1961; Karnovsky and Roots 1964; Tsuji 1974). Biochemical determinations showed that about 90% of the acetylcholinesterase activity originally present in the Triton extract were still detectable in the films. The relationship of the formation of cuprous thiocholine iodide in the case of the methods of Lewis (1961) or Tsuji (1974) and of cupric ferrocyanide at the reaction of Karnovsky and Roots (1964) to either enzyme concentration or incubation time were tested in detail. The results showed that for the method of Tsuji and, with some restrictions, also for the method of Karnovsky and Roots a linearity exists in these two respects. In the case of the Lewis technique, an approximate linearity between the amount of reaction product and incubation time could only be found from 90 min onward, but no linearity was detected in relation to the enzyme concentration. At low enzyme concentrations, too little white precipitate was formed in comparison to higher ones. Therefore it is suggested that this technique, as compared to the methods of Tsuji and Karnovsky and Roots, probably is less suitable as a quantitative cytochemical method.