Journal of biochemical and biophysical methods最新文献

{"title":"Detection of an abasic site in RNA with stem-loop DNA beacons: Application to an activity assay for Ricin Toxin A-Chain","authors":"Setu Roday ,&nbsp;Matthew B. Sturm,&nbsp;Dukagjin M. Blakaj,&nbsp;Vern L. Schramm","doi":"10.1016/j.jprot.2007.12.010","DOIUrl":"10.1016/j.jprot.2007.12.010","url":null,"abstract":"<div><p>The catalytic ability of Ricin Toxin A-Chain (RTA) to create an abasic site in a 14-mer stem-tetraloop RNA is exploited for its detection. RTA catalyzes the hydrolysis of the <em>N</em>-glycosidic bond of a specific adenosine in the G<strong><u>A</u></strong>GA tetraloop of stem-loop RNA. Thus, a 14-mer stem-loop RNA substrate containing an intact “G<strong><u>A</u></strong>GA” sequence can be discriminated from the product containing an abasic “G<strong><u>ab</u></strong>GA” sequence by hybridization with a 14-mer DNA stem-loop probe sequence and following the fluorescent response of the heteroduplexes. Three DNA beacon probe designs are described. Beacon 1 probe is a stem-loop structure and has a fluorophore and a quencher covalently linked to the 5′- and 3′-ends. In this format the probe–substrate heteroduplex gives a fluorescent signal while the probe–product one remains quenched. Beacon 2 is a modified version of 1 and incorporates a pyrene deoxynucleoside for recognition of the abasic site. In this format both the substrate and product heteroduplexes give a fluorescent response. Beacon 3 utilizes a design where the fluorophore is on the substrate RNA sequence at its 5′-end while the quencher is on the probe DNA sequence at its 3′-end. In this format the fluorescence of the substrate–probe heteroduplex is quenched while that of the product–probe one is enhanced. The lower limit of detection with beacons is 14 ng/mL of RTA.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 6","pages":"Pages 945-953"},"PeriodicalIF":0.0,"publicationDate":"2008-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jprot.2007.12.010","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27265962","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Analytical approach for selecting normalizing genes from a cDNA microarray platform to be used in q-RT-PCR assays: A cnidarian case study","authors":"Mauricio Rodriguez-Lanetty ,&nbsp;Wendy S. Phillips ,&nbsp;Sophie Dove ,&nbsp;Ove Hoegh-Guldberg ,&nbsp;Virginia M. Weis","doi":"10.1016/j.jbbm.2007.08.005","DOIUrl":"10.1016/j.jbbm.2007.08.005","url":null,"abstract":"<div><p>Research in gene function using Quantitative Reverse Transcription PCR (q-RT-PCR) and microarray approaches are emerging and just about to explode in the field of coral and cnidarian biology. These approaches are showing the great potential to significantly advance our understanding of how corals respond to abiotic and biotic stresses, and how host cnidarians/dinoflagellates symbioses are maintained and regulated. With these genomic advances, however, new analytical challenges are also emerging, such as the normalization of gene expression data derived from q-RT-PCR. In this study, an effective analytical method is introduced to identify candidate housekeeping genes (HKG) from a sea anemone (<em>Anthopleura elegantissima</em>) cDNA microarray platform that can be used as internal control genes to normalize q-RT-PCR gene expression data. It is shown that the identified HKGs were stable among the experimental conditions tested in this study. The three most stables genes identified, in term of gene expression, were beta-actin, ribosomal protein L12, and a Poly(a) binding protein. The applications of these HKGs in other cnidarian systems are further discussed.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 6","pages":"Pages 985-991"},"PeriodicalIF":0.0,"publicationDate":"2008-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jbbm.2007.08.005","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27026287","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Methodology to label mixed carbohydrate components by APTS","authors":"Annamária Bui ,&nbsp;Béla Kocsis ,&nbsp;Ferenc Kilár","doi":"10.1016/j.jprot.2008.01.009","DOIUrl":"10.1016/j.jprot.2008.01.009","url":null,"abstract":"<div><p>Labelling of carbohydrates with fluorescent dyes is of importance in the analysis of complex oligo- or polysaccharides. The complexity of the hydrolization mixtures of the oligosaccharides can be studied with separation methods after labelling. However, the procedures might provide unexpected phenomena. The experimental circumstances for labelling carbohydrates with 8-aminopyrene-1,3,6-trisulfonic acid were studied and the labelling efficiency of mono and oligosaccharides present in endotoxins was followed by capillary electrophoresis using LIF detection. Significant differences were observed in the labelling of the different sugar molecules, and the lowest reactivity with the dye was observed in the cases of amino-sugars. The results provide a basis for studies in determining the structures of the core parts and the O-chains of bacterial endotoxins.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 6","pages":"Pages 1313-1316"},"PeriodicalIF":0.0,"publicationDate":"2008-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jprot.2008.01.009","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27300015","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biophysical characterization of double-stranded oligonucleotides using ETBR and isothermal fluorescence spectroscopy: Implication for SNP genotyping","authors":"Pradip Bhattacharya,&nbsp;Swarkar Sharma,&nbsp;Sailesh Gochhait,&nbsp;Rameshwar N.K. Bamezai","doi":"10.1016/j.jprot.2007.10.001","DOIUrl":"10.1016/j.jprot.2007.10.001","url":null,"abstract":"<div><p>The UV-absorption, fluorescence and CD spectra of aps 23 bp oligoduplexes were performed for potential diagnostic purpose. These oligonucleotide sequences were mimicked from natural mutations (mitochondrial genome) of human population (unpublished). This work was designed on the basis of hybridization of non-self complementary oligoduplexes (aps) containing no mismatch, one-mismatch and two-mismatches. Since melting temperature™ is dependent on concentration of the oligoduplex, various concentrations were used in this study protocol. The thermal spectra profiles (UV absorbance and fluorescence) of these oligoduplexes (aps) are different for a particular concentration, and can be implicated for mutations. −<!--> <!-->d<em>F</em>/d<em>T</em> (or d<em>A</em>/d<em>T</em>) vs <em>T</em>, ln<em>K</em> (or <em>R</em>ln<em>K</em>) vs <em>T_M</em>, Δ<em>G</em> vs <em>T_M</em>, Δ<em>S</em> vs <em>T_M</em> and Δ<em>H</em> vs <em>T_M</em> are also variable for those sequences. All these thermodynamic data were calculated from absorbance (at 260 nm) data. On the contrary to the 23 bp oligoduplexes (aps), the PCR products of 97 bp and 256 bp length were genotyped with ETBR (excitation 530 nm, emission 600 nm) fluorimetrically. But our attempts to genotype these PCR sequences with isothermal UV absorbance spectroscopy were unsuccessful. Isothermal UV absorbance spectra has a limitation of sequence length. However, the structural conformation (all B-type) of the oligoduplexes (aps) was determined using CD. The minor discrepancy in CD spectra of these oligoduplexes are not significant for mutational analysis. 97 bp nested PCR product was an amplicon having either GcT or AcC mutation of mitochondria of normal human population, whereas 256 bp PCR product was an amplicon of human BRCA2 gene (NCBI Accession No. <span>AY151039</span><svg><path></path></svg>) of chromosome 13 having either A or G mutation at position −<!--> <!-->26.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 6","pages":"Pages 1163-1173"},"PeriodicalIF":0.0,"publicationDate":"2008-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jprot.2007.10.001","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27208485","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cyanine dye–protein interactions: Looking for fluorescent probes for amyloid structures","authors":"K.D. Volkova ,&nbsp;V.B. Kovalska ,&nbsp;A.O. Balanda ,&nbsp;R.J. Vermeij ,&nbsp;V. Subramaniam ,&nbsp;Yu. L. Slominskii ,&nbsp;S.M. Yarmoluk","doi":"10.1016/j.jbbm.2007.03.008","DOIUrl":"10.1016/j.jbbm.2007.03.008","url":null,"abstract":"<div><p>We ascertained the ability to detect fibrillar β-lactoglobulin (BLG) of a series of mono-, tri-, penta-, and heptamethinecyanines based on benzothiazole and benzimidazole heterocycles, and of benzothiazole squaraine. Fluorescence properties of these cyanine dyes were measured in the unbound state and in the presence of monomeric and fibrillar BLG and compared with those for the commercially available benzothiazole dye Thioflavin T. The correlation between the chemical nature of the dye molecules and the ability of dyes to bind aggregated proteins was established. We found that meso-substituted cyanines with amino substituents in heterocycle in contrast to the corresponding unsubstituted dyes have a binding preference to fibrillar BLG and a noticeable fluorescence response in the presence of the aggregated protein. For the squaraines and benzimidazole penthamethinecyanines studied, fluorescence emission increased both in the presence of native and fibrillar protein. The trimethinecyanines T-49 and SH-516 exhibit specifically increased fluorescence in the presence of fibrillar BLG. These dyes demonstrated the same or higher emission intensity and selectivity to aggregated BLG as Thioflavin T, and are proposed for application in selective fluorescent detection of aggregated proteins.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 5","pages":"Pages 727-733"},"PeriodicalIF":0.0,"publicationDate":"2007-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jbbm.2007.03.008","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"26693967","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Application of Fourier linear spectral analyses to the characterization of smooth muscle contractile signals","authors":"Mario Díaz","doi":"10.1016/j.jbbm.2007.03.007","DOIUrl":"10.1016/j.jbbm.2007.03.007","url":null,"abstract":"<div><p>In intestinal smooth muscle, peristaltic activity emerges from a depolarization wave generated by Cajal's interstitial cells that travels longitudinally and transversally towards adjacent smooth muscle. The electrophysiological diversity of cell populations involved in the generation and transmission of excitation between nerve and muscle cells, as well as the specialization in the excitation–contraction coupling of smooth muscle cells, makes it difficult to extrapolate individual cell responses to overall peristaltic activity. It is conceivable intestinal contractile activity as the macroscopic output from a multicellular system in time and frequency domains. Given that contractile signals are usually linear and stationary, application of frequency analyses using the discrete Fourier transform allows the accurate definition of amplitudes and phases of harmonic components in the frequency spectrum of contractile activity records, as well as the power spectrum of the signal. In addition, by using the short-time Fourier transform it is also possible to obtain the spectrogram of contractile signals, which allows the identification of non-stationary events. Often, the combined usage of these types of analyses together with specific pharmacological and molecular biology tools is sufficient to unveil the cellular and molecular locus of action of modulators of peristaltism, including hormones and different natural and synthetic compounds.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 5","pages":"Pages 803-808"},"PeriodicalIF":0.0,"publicationDate":"2007-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jbbm.2007.03.007","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"26693965","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A convenient and efficient protocol for isolating high-quality RNA from latex of Hevea brasiliensis (para rubber tree)","authors":"Chaorong Tang,&nbsp;Jiyan Qi,&nbsp;Heping Li,&nbsp;Cunliang Zhang,&nbsp;Yuekun Wang","doi":"10.1016/j.jbbm.2007.04.002","DOIUrl":"10.1016/j.jbbm.2007.04.002","url":null,"abstract":"<div><p>Isolating high-quality RNA from latex of <em>H. brasiliensis</em> is a prerequisite to elucidating the molecular mechanisms of rubber biosynthesis and its regulation. Here, an improved protocol was developed for latex collection, transportation, storage, and RNA isolation. Compared with existing ones, our protocol eliminated liquid nitrogen for latex collection and subsequent low-temperature (−<!--> <!-->70 °C) condition for latex storage, making it more convenient and feasible when latex was collected in remote sampling sites, and latex storage and RNA isolation were conducted in poorly-equipped laboratories. Different methods (UV absorbance scans, denaturing gel electrophoresis, autoradiograph monitoring of cDNA synthesis) were used to confirm the high quality of the RNA prepared with this protocol, whose usefulness was further verified by several practical applications, including construction of one high-quality cDNA library, cloning of the full-length cDNAs of 3 novel <em>Hevea</em> sucrose transporter genes, and semi-quantitative RT-PCR analysis of two rubber-biosynthesis essential genes and one sucrose transporter gene.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 5","pages":"Pages 749-754"},"PeriodicalIF":0.0,"publicationDate":"2007-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jbbm.2007.04.002","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"26713034","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The use of Quantitative Agarose Gel Electrophoresis for rapid analysis of the integrity of protein–DNA complexes","authors":"Nicholas L. Adkins ,&nbsp;J. Adam. Hall ,&nbsp;Philippe T. Georgel","doi":"10.1016/j.jbbm.2007.03.006","DOIUrl":"10.1016/j.jbbm.2007.03.006","url":null,"abstract":"<div><p>Recent biochemical studies evaluated the affinity of histones to DNA in the context of nucleosome core particle (NCP). These have indicated a concentration-dependence for nucleosome stability. However, when studying chromatin the preferred templates are nucleosome arrays (NA) and not the NCP. Biochemical methods are poorly suited for structural analysis of chromatin. To overcome that technical hindrance, and investigate the effect of concentration on stability of the histone–DNA interactions, we have applied the multigel Quantitative Agarose Gel Electrophoresis (QAGE) method to in vitro-assembled nucleosomal arrays. The results demonstrated the method to be extremely valuable for the evaluation of the effect of low concentration on NA. However, QAGE is a fairly time-demanding and complex method. To maximize the efficiency of use of this technology, we devised a protocol that allowed for multiple sets of templates to be analyzed simultaneously. Briefly, samples can be loaded at regular intervals and analyzed individually for their molecular composition. The technique presented in this study describes the calibration steps and proof of concept necessary to validate the use of multiple loading of multigel to evaluate the composition of nucleosomal arrays as a function of concentration.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 5","pages":"Pages 721-726"},"PeriodicalIF":0.0,"publicationDate":"2007-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jbbm.2007.03.006","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"26807004","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Novel fluorescence detection technique for non-contact temperature sensing in microchip PCR","authors":"Sudip Mondal,&nbsp;V. Venkataraman","doi":"10.1016/j.jbbm.2007.05.001","DOIUrl":"10.1016/j.jbbm.2007.05.001","url":null,"abstract":"<div><p>DNA amplification using Polymerase Chain Reaction (PCR) in a small volume is used in Lab-on-a-chip systems involving DNA manipulation. For few microliters of volume of liquid, it becomes difficult to measure and monitor the thermal profile accurately and reproducibly, which is an essential requirement for successful amplification. Conventional temperature sensors are either not biocompatible or too large and hence positioned away from the liquid leading to calibration errors. In this work we present a fluorescence based detection technique that is completely biocompatible and measures directly the liquid temperature. PCR is demonstrated in a 3 μL silicon-glass microfabricated device using non-contact induction heating whose temperature is controlled using fluorescence feedback from SYBR green I dye molecules intercalated within sensor DNA. The performance is compared with temperature feedback using a thermocouple sensor. Melting curve followed by gel electrophoresis is used to confirm product specificity after the PCR cycles.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 5","pages":"Pages 773-777"},"PeriodicalIF":0.0,"publicationDate":"2007-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jbbm.2007.05.001","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"26778839","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"DNA immobilization chemical interference due to aggregates study by Dip and Drop approach","authors":"Ram Ajore ,&nbsp;Rakesh Kumar ,&nbsp;Inderpreet Kaur ,&nbsp;R.C. Sobti ,&nbsp;Lalit M. Bharadwaj","doi":"10.1016/j.jbbm.2007.05.006","DOIUrl":"10.1016/j.jbbm.2007.05.006","url":null,"abstract":"<div><p>In the present manuscript, we report the studies and observations for chemical interference due to aggregates formation during covalent immobilization of thiolated λ-DNA between gold microelectrodes. Dip and Drop approaches were employed to study DNA immobilization using thiolated oligos (oligoA 5′ GGGCGGCGACCT 3′ and oligoB 5′ AGGTCGCCGCCC 3′). As a result of aggregation, less interference was observed in Dip approach as compared to Drop approach. Atomic Force Microscopy (AFM) analysis of piranha treated gold surface revealed 47.5% increase in height roughness, contributing in interference by creating active sites. Cyclic voltammetry (CV) studies ascertain the multitude of adsorption states existing in long strand of DNA on surface. Surface coverage was found to be ∼ 72% (1.35<!--> <!-->×<!--> <!-->10¹⁰ molecules/cm²), and ∼ 42% (7.89<!--> <!-->×<!--> <!-->10⁹ molecules/ cm²) in Dip and Drop approach, respectively. Dip approach can be used as a measure to minimize interference due to aggregation.</p></div>","PeriodicalId":15257,"journal":{"name":"Journal of biochemical and biophysical methods","volume":"70 5","pages":"Pages 779-785"},"PeriodicalIF":0.0,"publicationDate":"2007-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.jbbm.2007.05.006","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"26801805","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}