bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rapid mitochondrial DNA typing using restriction enzyme digestion of polymerase chain reaction amplications followed by capillary electrophoresis separation with laser‐induced fluorescence detection
Authors: Butler John M. , Wilson Mark R. , Reeder Dennis J. 
Published: May 2005

AbstractThe polymorphic control region of mitochondrial DNA (mtDNA) is becoming more commonly used in forensic applications to differentiate among individuals in a population. Two hypervariable regions (HV1 and HV2) are often sequenced following amplification of the mtDNA via the polymerase chain reaction (PCR). More rapid screening assays would reduce both the effort and the expenses of comparing two samples. A methodology has been developed that first uses restriction endonuclease digestion of the PCR‐amplified mtDNA using RsaI and Mn/I and then capillary electrophoresis (CE) to separate and size the PCR‐RFLP fragments. This rapid procedure offers an alternative method for screening of polymorphisms in amplified mtDNA samples. In addition, the presence of a T→C transition at position 16189, which gives rise to the so‐called “C‐stretch” in HV1, may be predicted from the presence of nonspecific PCR products in the CE results.
Oligonucleotide DNA fingerprinting optimized to determine parentage in three beetle species
Authors: Benken Theodor , Müller Josef K. , Peschke Klaus 
Published: May 2005

AbstractOligonucleotide fingerprinting was performed on three beetle species from different families (Coleoptera: Staphylinidae: Aleochara curtula; Silphidae: Nicrophorus vespilloides; Tenebrionidae: Blaps lethifera) to obtain detailed information on parentage in mating systems. We report variations in the number of hybridizing fragments for the different species depending on the combinations of probes and restriction enzymes used. In addition to conventional multilocus fingerprint patterns, we established a single locus system for A. curtula (GTG5; HinfI) and described an oligolocus system in N. vespilloides (GATA4; HaeIII).
Detection of p53 point mutations by single strand conformation polymorphism: Analysis by capillary electrophoresis
Authors: Atha Donald H. , Wenz H.‐Michael , Morehead Harvard , Tian Jing , O'Connell Catherine D. 
Published: May 2005

AbstractWe have analyzed five p53 single point mutations by single strand conformation polymorphism using capillary electrohoresis (CE‐SSCP) and have compared these measurements to measurements obtained by slab gel electrophoresis (SG‐SSCP). PCR primers were used for amplification of specific exons for mutation detection. 5′ Primers were labeled with FAM (5‐carboxyfluorescein) and 3′ primers were labeled with JOE (2′,7′‐dimethoxy‐4′,5′‐dichloro‐6‐carboxyfluorescein). CE‐SSCP was performed using the Perkin Elmer ABI PRISM™ 310 Genetic Analyzer with GeneScan™ Software and the Beckman P/ACE™ 5510 CE equipped for laser‐induced fluorescence detection. Although the shifts in migration times for the p53 mutations relative to the corresponding wild‐type strands could be successfully detected by either SG or CE analysis, the individual electrophoresis run times were about tenfold faster and more automated with capillary electrophoresis. The CE‐SSCP measurements were performed at temperatures ranging from 10 to 60°C on a prototype instrument. For mutations measured at ambient temperature (25°C), characteristic shift in direction and magnitude were observed in the migration times to both strands of all mutations relative to the wild type. This demonstrated the ability of CE at ambient temperature to resolve these mutations. However, the magnitute and direction of shifts in migration time varied with temperature in a discrete pattern for each mutation and resulted in a temperature‐specific profile for each mutation. This demonstrated that extended temperature control will be an important advantage in resolving single point mutations by CE‐SSCP. In addition, by using CE, discrete intra‐strand isoforms could be easily observed at different temperatures. The combination of mutation‐specific temperature profiling and analysis of isoforms by CE‐SSCP should be of help to the diagnostic community in the detection of genetic mutations.
Enhanced detection sensitivity of “fluorescence reduction” by shifting the analyte absorbance spectrum and use of a fluorescent paper with higher signal/noise ratio
Authors: Yarmola Elena , Chen Nong , Yi David , Chrambach Andreas 
Published: May 2005

AbstractNonfluorescing protein bands can be detected by the fluorescence optics of the commerical gel electrophoresis apparatus with automated scanning of the migration path (HPGE‐1000, LabIntelligence, Belmont CA), taking advantage of the decrease of emission from a fluorescent paper placed below the gel by the absorbance of proteins (“fluorescence reduction”). That decrease of fluorescence gives rise to an inverted protein peak. Nonfluorescent colorless proteins appear to reduce the intensity of light emitted from the fluorescent paper due to absorbance of incident and emitted light. When the absorbance spectrum only slightly overlaps with the excitation and emission spectra of the fluorescent paper, that reduction is weak, and detection sensitivity in that application is consequently only 1/30 of that of fluorescent proteins. By contrast, when the protein is colored so that its absorbance spectrum overlaps widely with the excitation and emission spectra of the fluorescent paper, the sensitivity of “fluorescence reduction” equals 1/4 to 1/5 of that obtained for fluorescent proteins. Bands detected by “fluorescence reduction” provide a quantitative measure of protein load and mobility. The area of the inverted bands is proportional to protein loads up to 16 μg/lane of the gel tray. A theory of “fluorescence reduction” is presented which accounts for the existence of a linear relationship between band area and load.
Viscosity‐adjustable block copolymer for DNA separation by capillary electrophoresis
Authors: Wu Chunhung , Liu Tianbo , Chu Benjamin 
Published: May 2005

AbstractThe viscosity‐adjustable property of F127 block copolymer PEO99PPO69PEO99, PEO and PPO being poly (ethylene oxide) and poly(propylene oxide), respectively, was found to be useful for the development of automated capillary electrophoresis (CE). The polymer solution can form a gel‐like structure with sieving ability and can also serve as a dynamic coating material, thereby effectively suppressing the electroosmotic flow induced by the ionization of the silanol group on the quartz capillary inner wall. When applied to CE as a separation medium, F127 block copolymer can provide the advantages of high separation resolution, easy injection and replacement of the triblock copolymer solution and convenient capillary column treatment. High reproducibility of DNA electrophoretic migration time in CE by replenishing F127 solution in acid‐washed capillary tubings can be achieved. The relative standard deviation of the DNA migration time is less than 2%. In the investigation of F127 concentration and temperature effects on the performance of DNA separation in CE, we have found that the DNA electrophoretic migration behavior in the F127 gel‐like solution cannot be described by any one of the existing models.
Characterization of high molecular mass linear polyacrylamide powder prepared by emulsion polymerization as a replaceable polymer matrix for DNA sequencing by capillary electrophoresis
Authors: Goetzinger Wolfgang , Kotler Lev , Carrilho Emanuel , Ruiz‐Martinez Marie C. , Salas‐Solano Oscar , Karger Barry L. 
Published: May 2005

AbstractIn a previous paper, a 2% w/w replaceable high molecular mass linear polyacrylamide solution (high molecular mass LPA) was used to achieve long readlengths for DNA sequencing by capillary electrophoresis (E. Carrilho et al. Anal. Chem. 1996, 68, 3305–3313). In that work, the polymer was prepared by polymerization in water at 6% w/w, followed by dilution to 2% w/w. In this study, an improved method for preparation of high molecular mass LPA was developed, based on inverse emulsion polymerization. With this polymerization procedure, the LPA results in a molecular mass of approximately 9 MDa with characteristics of a fine powder of high purity and practically unlimited shelf life. Using size exclusion chromatography (SEC) and viscosity measure ments to characterize the polymer, good batch‐to‐batch reproducibility was found. It was observed that the viscous polymer solutions made from these high molecular mass polymers require careful preparation and handling because the method of dissolution could affect the molecular mass distribution and the resultant separation of DNA components. Solution containing 2% w/w of LPA made by emulsion polymerization were simple to prepare resulting in excellent performance as a replaceable matrix for DNA sequencing by capillary electrophoresis. The viscosity of the polymer decreased exponentially when pressure was applied. allowing easy replacement from a capillary using a syringe. With a properly prepared matrix, a read‐length on morethan 1000 bases in 80 min with an accuracy better than 97%, and better than 99% for the first 800 bases, could be achieved.
Selenium speciation by interfacing capillary electrophoresis with inductively coupled plasma‐mass spectrometry
Authors: Michalke Bernhard , Schramel Peter 
Published: May 2005

AbstractThe high resolution potential of capillary electrophoresis (CE) makes CE techniques valuable for separations of selenium species. Further, inductively coupled plasma‐mass spectrometry (ICP‐MS) affords element‐specific multi‐element detection, providing extremely low detection limits. The combination of CE with ICP‐MS promises to become a powerful tool for metal speciation. Therefore, an on‐line hyphenation of CE with ICP‐MS, which was developed earlier (Michalke, B., Schramel, P., Fresenius' J. Anal. Chem. 1997, 257, 594‐599), was modified and applied to selenium speciation. For this reason, capillary zone electrophoresis (CZE) methods were developed, providing the possibility to analyze six Se species of interest in one run: Se (IV), Se (VI), selenium carrying glutathione (GSSeSG), selenomethionine (SeM), selenocystine (SeC), and selenocystamine (SeCM). The final CE method used an alkaline background electrolyte (Na2CO3/NaOH) with separation differentiated from the detection step during hyphenation. This resulted in short separation times (10 min) and a subsequent detection step of 100s. The Se species were sufficiently separated from each other and appeared at 7s (SeCM), 16s (Se (VI)), 22s (SeC), 27s (Se (IV)), 35s (SeM) and 56s (GSSeSG) during the detection step. Detection limits were calculated as 10 or 20 μg Se/L for inorganic Se species and 35–50 μg Se/L for organic Se species.
Quantitative trace analysis of L‐ascorbic acid in human body fluids by on‐line combination of capillary isotachophoresis and zone electrophoresis
Authors: Procházková Andrea , Křivánková Ludmila , Boček Petr 
Published: May 2005

AbstractOn‐line combination of capillary isotachophoresis and zone electrophoresis performed in two coupled capillaries (ITP‐CZE) is used for the trace analysis of L‐ascoribic acid in human serum, urine and stomach fluid. At the ITP stage, anionic sample components are separated into individual zones and macrocomponents are detected and driven out of the migration path. In the CZE stage, only a small segment of the sample zones containing L‐ascorbic acid is analyzed. High sensitivity of this hyphenated method (limit of detection, 0.09–0.15 mg/L), low sample volume consumption (2 μL), and acceptable reproducibility of the results (relative standard deviation, 8%) in the concentration range 0.1–15 mg/L demonstrate that the method is applicable for the study of the relation between the content of L‐ascorbic acid in body fluids and the state of health of a person, in which lower amounts of L‐ascorbic acid than the normal levels (i.e., 501–15.1 mg/L in human serum and 12.5–26.8 mg/L in urine) are expected. Possible interferences of other components of the body fluids are excluded by good correlation of the results obtained by the ITP‐CZE method and a routine colorimetric method.
Analysis of changes in acute‐phase plasma proteins in an acute inflammatory response and in rheumatoid arthritis using two‐dimensional gel electrophoresis
Authors: Doherty Niall S. , Littman Bruce H. , Reilly Karen , Swindell Archie C. , Buss Jane M. , Anderson N. Leigh 
Published: May 2005

AbstractTwo‐dimensional (2‐D) gel analysis was used to examine differences in the levels of 19 plasma proteins: before and after an acute inflammatory reaction (parenteral typhoid vaccination) in normal subjects, between rheumatoid arthritis (RA) patients and normals and in RA patients treated with tenidap (120 mg) and piroxicam (20 mg). Typhoid vaccination increased levels of SAA, haptoglobin α1, haptoglobin α2, haptoglobin β and α1‐anti‐chymotrypsin but decreased transthyretin and apolipoprotein E. In RA patients, serum amyloid A (SAA), haptoglobin α2, haptoglobin β, α1‐antichymotrypsin and C3 proactivator levels were elevated while apolipoprotein A‐I, apolipoprotein A‐IV, transthyretin, Gc‐globulin, α2‐HS glycoprotein, α2‐macroglobulin and α1‐B glycoprotein levels were decreased, compared to normals. Compared to piroxicam, tenidap lowered levels of α1‐antiprotease and SAA but raised the levels of transthyretin, Gc‐globulin, α2‐HS‐glycoprotein and α2‐macroglobulin in RA patients. C‐reactive protein (CRP) could not be quantified on 2‐D gels but, When measured by rate nephelometry, levels were reduced after treatment with tenidap compared to piroxicam. The general pattern of the acute phase protein response to an acute inflammatory response to typhoid vaccination is similar to that in the chronic inflammatory condition, RA. The impact of tenidap on both positive and negative acute‐phase proteins in RA patients could clearly be distinguished from that of piroxicam.
Dynamic complexation of solutes in capillary electrophoresis
Authors: Bowser Michael T. , Chen David D. Y. 
Published: May 2005

AbstractThe analyte migration behavior in any chemical separation system can be described using a single equation that unifies all areas of separation science. This equation can be used in capillary electrophoresis (CE) to design separation systems, and to study interactions between analytes and additives. By using individual capacity factors for each analyte species present in the system, and with the knowledge of the characteristics of each interaction, one can predict the analyte migration behavior in complicated CE systems, including systems with multiple 1:1 interactions and/or higher order interactions.
On‐line competitive immunoassay based on capillary electrophoresis applied to monitoring insulin secretion from single islets of Langerhans
Authors: Tao Li , Aspinwall Craig A. , Kennedy Robert T. 
Published: May 2005

AbstractAn automated on‐line competitive immunoassay based on capillary electrophoresis (CE) was utilized to monitor secretion of insulin from single islets of Langerhans stimulated by glucose and tolbutamide. In the instrument, fluorescein isothiocyanate‐labeled insulin (FITC‐insulin), monoclonal anti‐insulin and perifusate of single islets were mixed on‐line while islets were exposed to different levels of glucose and tolbutamide. Insulin released from single islets competed with FITC‐insulin for antibody binding sites. Therefore, the amounts of bound and free FITC‐insulin were modulated by insulin released from islets. The bound and the free FITC‐insulin were separated by CE every 3 s and the bound over free ratio (B/F) was measured. Insulin levels were obtained by comparing B/F with calibration curves obtained under the same conditions except that the islet perfusate was replaced with various concentrations of insulin. Patterns of insulin secretion stimulated by glucose and tolbutamide observed were comparable to what has been seen previously using radioimmunoassay or enzyme‐linked immunoassay. This on‐line competitive immunoassay system provided a fast and direct way to measure insulin release from single islets. The effects of temperature on antibody‐antigen reaction rate and binding equilibrium were also studied.
Affinity capillary electrophoresis investigation of an epitope on human immunodeficiency virus recognized by a monoclonal antibody
Authors: Qian Xiao‐Hong , Tomer Kenneth B. 
Published: May 2005

AbstractAffinity capillary electrophoresis (ACE) has been used to investigate the epitope on the human immunodeficiency virus (HIV) core protein p24 recognized by the monoclonal antibody (mAb) 13‐102‐100. The affinity of a series of peptides with N‐ and C‐terminal truncations of the epitope sequence determined by mass spectrometry was studied. The peak area change assay was used for the study of the interactions of the mAb with those peptides, exhibiting tight binding to the mAb, and the migration time shift assay was used to probe the relative affinities of peptides showing weak binding to the mAb. The experimental results show that the monoclonal antibody 13‐102‐100 recognizes the peptide VHPVHAGPIAP with highest affinity. Smaller peptides incorporating only part of the epitope, however, are recognized to some extent in the ACE experiments.
Affinity capillary electrophoresis employing immobilized glycosaminoglycan to resolve heparin‐binding peptides
Authors: VanderNoot Victoria A. , Hileman Ronald E. , Dordick Jonathan S. , Linhardt Robert J. 
Published: May 2005

AbstractA new capillary electrophoresis technique has been developed for the affinity resolution of synthetic heparin‐binding peptides using an immobilized glycosaminoglycan. Heparin and heparan sulfate were immobilized onto fused silica capillaries using biotin‐neutravidin conjugation. These capillaries exhibited markedly reduced electroosmotic flow and were able to distinguish peptides based on the heparin binding domain of acidic fibroblast growth factor (residues 125–144, GLKKNGSCKRGPRTHYGQKA) that differed only in the stereochemistry of the proline amino acid residue. The peptide based on the native sequence was retarded compared to the peptide having unnatural stereochemistry, consistent with its stronger interaction for immobilized glycosaminoglycan. Improved resolution is also obtained for additional arginine and lysine containing heparin‐binding peptides.
Cloning of prokaryotic genomes in yeast artificial chromosomes: Application to the population genetics of Pseudomonas aeruginosa
Authors: Heuer Thomas , Bürger Christiane , Maaß Giinter , Tümmler Burkhard 
Published: May 2005

AbstractYeast artificial chromosomes (YACs) can accommodate large inserts and hence should be attractive tools for intra‐ and interspecies comparisons of bacterial genomes. YAC libraries were constructed from size‐selected partial digests of human and Pseudomonas aeruginosa PAO DNA and SpeI‐restricted PAO DNA. Whereas YACs from human DNA had an average size of 350 kilobase pairs (kbp), a P. aeruginosa sequence larger than 120 kbp was absent or truncated in the eukaryotic host. Coligation occurred for YACs smaller than 40 kbp, but stable YACs with 40–120 kbp large inserts of P. aeruginosa DNA were obtained in high yield. SpeI‐restricted chromosomes from 97 P. aeruginosa strains representing 47 genotypes were hybridized with stable YACs from three equidistant regions of the PAO genome. The low complexity of hybridizing bands demonstrated that the analyzed 100 kbp sequence contigs were stably maintained in most P. aeruginosa isolates from both disease and environmental habitats.
Differential genome analysis of bacteria by genomic subtractive hybridization and pulsed field gel electrophoresis
Authors: Schmidt Karen D. , Schmidt‐Rose Thomas , Römling Ute , Tümmler Burkhard 
Published: May 2005

AbstractA comprehensive analysis of the differences between the genomes of two closely related bacterial strains should give insight into the molecular basis of their individual phenotypic and genotypic characteristics. Here we present an integrative approach including two different strategies for the thorough investigation of genomic divergence. We have combined two techniques including genomic subtractive hybridization and comparative genome mapping by pulsed field gel electrophoresis (PFGE) techniques. The subtractive method for which a protocol is given herein results in the production of a library of specific DNA sequence tags present only in one strain, while the construction of macrorestriction maps of the bacterial chromosomes yields data about the overall genome organization and the arrangement and distance of gene loci. Comparison of the physical and genetic maps and determination of the map positions of the strain‐specific DNA sequences reveals gross chromosomal modifications, insertions or deletions of additional genetic material, and transpositional events. The further investigation of the strain‐specific regions yields information about the nature and origin of the acquired DNA and their influence on the evolution of the individual bacterial genome. The two methods were applied to differential genome analysis of clonal divergence in Pseudomonas aeruginosa choosing two clone C isolates from diverse habitats.
Low molecular weight proteins: A challenge for post‐genomic research
Authors: Rudd Kenneth E. , Humphery‐Smith Ian , Wasinger Valerie C. , Bairoch Amos 
Published: May 2005

AbstractThe EcoGene project involves the examination of Escherichia coli K‐12 DNA sequences and accompanying annotation in the public databases in order to refine the representation and prediction of the entire set of E. coli K‐12 chromosomally encoded protein sequences. The results of this ongoing effort have been deposited in the SWISSPROT protein sequence database as sequencing of the E. coli genome has progressed to completion in recent years. Through this continuing research, we have discovered that the prediction of low molecular weight (small) proteins, arbitrarily defined as protein sequences ≤ 150 amino acids (aa) in length, is problematic and requires special attention. We describe the small protein subset of EcoGene and the approach used to derive this subset from the complete E. coli genome sequence and database annotations. These E. coli proteins have helped to identify new small genes in other organisms and to identify conserved residues (motifs) using database searches and multiple alignments. Two thirds of the E. coli small proteins have not been characterized experimentally. The careful application of computer and laboratory methods to the analysis of small proteins is needed for accurate prediction, verification and characterization. The problem of accurate protein sequence identification is not limited to small proteins or to E. coli; these problems are encountered to varying degrees throughout all sequence databases.
Comparison of the genome organization of pathogenic Neisseriae (minireview)
Authors: Bautsch Wilfried 
Published: May 2005

AbstractCurrent efforts to completely sequence the meningococcal and gonocococcal genomes raise the question whether the lessons learned from the sequenced strains may be safely extrapolated to other members of these species, or whether, in view of the fact that Neisseriae are highly recombinogenic and exhibit a high degree of horizontal intra‐ and interspecies genetic transfer, only clonespecific conclusions are valid. From the known physical and genetic maps of each of two gonococcal and meningococcal strains, it would appear that both species exhibit a species‐specific conservation in their genetic organization while the interspecies comparison revealed several rearrangements, although still with a high overall similarity. However, these data contrast with other evidence suggesting intra‐species rearrangements, such as the nonconserved I‐CeulI macrorestriction patterns of different meningococcal and other neisserial strains. Since I‐Ceul cuts within the 23S‐rRNA sequence, the restriction pattern should give reliable information on the distribution of rrn loci in the neisserial genomes. Further studies are warranted to answer these questions.