bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased microsatellite variability in Macaca mulatta compared to humans due to a large scale deletion/insertion event during primate evolution
Authors: Kayser Manfred , Nürnberg Peter , Bercovitch Fred , Nagy Marion , Roewer Lutz
Published: May 2005

Human (GATA)n microsatellites D12S66 and D12S67 could be successfully amplified by polymerase chain reaction (PCR) in various species of apes and monkeys. In 86 unrelated animals of the most intensively studied species Macaca mulatta we demonstrated five alleles at “D12S66” differing in size in increments of 4 bp (159–175 bp), whereas 17 alleles were observed at locus “D12S67”. The alleles of the latter locus are distributed in two separate groups with no alleles of intermediate size. Six alleles were found between 108–128 bp and 11 alleles between 181–249 bp. Mendelian inheritance of the codom‐inant alleles was proven by family studies. Sequencing of the “D12S67” locus revealed that the shorter alleles are characterized by a single perfect (GATA)n stretch whereas the longer alleles consist of two blocks of (GATA)n repeats separated by an intervening sequence of 9 bp. The composite structure of the longer alleles closely resembles that of the 12 human D12S67 alleles (229–273 bp). The enormous species variation in the fragment size range, with the smallest allele found in Macaca mulatta (108 bp) and the largest (364 bp) in Gorilla gorilla gorilla strongly indicates that D12S67 has been subjected to recurrent mutations over the course of primate evolution including a large deletion and/ or insertion event.
Arbitrarily amplified DNA in systematics and phylogenetics
Authors: Schierwater Bernd
Published: May 2005

Different polymerase chain reaction (PCR) based techniques which use single primers of arbitrary sequence have become prominent and powerful tools for taxonomic and genealogical studies. While the use of arbitrarily amplified DNA (AAD) markers for the identification of lower taxonomic units (e.g. at the species level) has been proven to be more or less straightforward, their potential usefulness in phylogenetic studies has not been sufficiently addressed yet. The main advantages of AAD markers include that they require only very small amounts of DNA, no sequence information of the taxa under investigation and the relative ease of generating high numbers of AAD markers in short time. On the other hand, the generation of AAD markers is susceptible to certain random and systematic errors, and for phylogenetic studies the taxonomic level at which informative markers can be generated is severely limited.
Genetic self‐management in a captive colony of mandrills (Mandrillus sphinx) as revealed by DNA minisatellite fingerprints
Authors: Wickings E. Jean
Published: May 2005

DNA fingerprints using three oligonucleotide probes, a (GTG)5 short tandem repeat sequence, and 2 corresponding to the core sequences of minisatellites 33.15 and 33.6, have been used to generate DNA fingerprints of a captive colony of mandrills (Mandrillus sphinx) housed at the Centre International de Recherches Médicales de Franceville (CIRMF), in order to monitor paternity. A colony of 6 males and 7 females was established in a 6 ha. rain‐forested enclosure in 1983. Band sharing scores indicated that none of the founders were related. By April 1994 the colony had increased to 84 individuals. Forty‐nine of the 77 births were first‐generation infants, a further 27 were second‐generation offspring, and one was a third‐generation mandrill. Five animals died during the study, and one was removed for husbandry reasons. Overall band sharing between individuals increased markedly and the power to discriminate between first‐ and second‐generation infants was reduced; the probability of two individuals sharing the same pattern increased 1000‐fold from 10−6 to 10−3. The dominant male in the group effectively guards access to all receptive females during the annual breeding season. Paternity analysis confirmed that the dominant male is the father of the great majority of offspring born during his term of dominance. Daughters born before 1991 to either of the two dominant males reached sexual maturity during his tenure. Incestual inbreeding occurred with both males, but only 25% of all possible cases resulted in the birth of a viable infant; one male fathered infants on 3 of 13 possible occasions and a second male on 2 of 7 occasions. Female avoidance strategies and shifts in male dominance in this closed mandrill colony appear to be timed in a way that reduces incest.
Immunoprinting reveals different genetic bases for (auto)immune diseases
Authors: Epplen Cornelia , Buitkamp Johannes , Rumpf Heike , D'Souza Markus , Epplen Jörg T.
Published: May 2005

The genetic basis of complex (auto)immune diseases has been studied for an ovine nematode infection, human rheumatoid arthritis (RA), early onset pauciarticular arthritis (EOPA) and multiple sclerosis (MS). Immunoprinting combines the powerful simplicity of polymerase chain reaction (PCR)‐based amplification of discrete, highly informative microsatellite loci with the principle of genetic associations. This approach has allowed us to define novel genetic risk factors in adult RA patient categories whereas EOPA forms in juveniles display other prominent genetic contributions. Differentially regulated tumor necrosis factor (TNF) expression may lead to a better understanding of the causal pathogenesis of EOPA while T cell receptor (TCR) gene polymorphisms appear crucial for RA manifestation in certain patient groups. Statistically significant marker associations have still to be defined for MS in larger panels of patient and control cohorts. The clinical course of the disease will probably have to be taken into account when associations with lymphokine levels are evaluated. In essence a convoluted myriad of negative and a few positive disease association data have been generated efficiently by immunoprinting. As expected, the interrelationships are truly complicated between the polymorphic genetic instances predisposing to autoimmune disease. Nevertheless, risk factors may be defined on an individualized basis by indirect gene diagnosis revealing predispositions and providing a more solid basis for differential diagnosis and treatment.
Parallel genome analysis by one‐ and two‐dimensional DNA fingerprinting in human gliomas
Authors: Nürnberg Peter , Marczinek Karola , Thiel Gundula , Hampe Jochen
Published: May 2005

The detection of DNA variation in cancers is an important step in elucidating the mechanism of tumorigenesis. Using the strategy of multipoint genome analysis we detected many differences between glioma‐derived and constitutional DNA by customary DNA fingerprinting with simple repetitive oligonucleotide probes. Amplification of the epidermal growth factor receptor (EGFR) gene has been found to be easily detectable as new or highly intensified bands in one‐dimensional (1‐D) DNA fingerprints of glioblastoma DNA generated with probes (GTG)5 or (GT)8. However, in most low‐grade astrocytomas, 1‐D DNA fingerprinting has failed to reveal any genomic abnormalities. In these cases a two‐dimensional (2‐D) technique was successfully employed that is based on size separation in neutral gels followed by sequence‐dependent separation in denaturing gradient gels and hybridization with several mini‐ and microsatellite core probes. The hundreds of spots visualized with this technique were used to detect subtle changes probably occurring as the initial steps of tumorigenesis in human gliomas. On average, five of the approximately 580 sports generated by probes CAC and 33.6 were found to be altered in tumor DNA; 80% of the alterations were spot losses, the rest being spot gains or amplifications. Computer‐based image analysis using an external lambda marker provided a stringent way to compare spot patterns generated by 2‐D DNA finger‐printing. In comparisons performed between typing patterns generated on the same gel, 99% of truly identical spots were confirmed by the sofware. In intergel comparisons 84% of identical spots were matched on the basis of the marker information alone.
DNA fingerprinting of Brassica juncea cultivars using microsatellite probes
Authors: Bhatia Sabhyata , Das Sandip , Jain Ajay , Lakshmikumaran Malathi
Published: May 2005

The genetic variability in the Brassica juncea cultivars was detected by employing in‐gel hybridization of restricted DNA to simple repetitive sequences such as (GATA)4, (GACA)4 and (CAC)5. The most informative probe/enzyme combination was (GATA)4/EcoRI, yielding highly polymorphic fingerprint patterns for the B. juncea cultivars. This technique was found to be dependable for establishing the variety specific patterns for most of the cultivars studied, a prerequisite for germplasm preservation. The results of the present study were compared with those reported in our earlier study in which random amplification of polymorphic DNA (RAPD) was used for assessing the genetic variability in the B. juncea cultivars.
Highly repetitive DNA sequence elements from Orseolia oryzae (Wood‐Mason) discriminate between the Indian isolates of the Asian rice gall midge and the paspalum midge
Authors: Ehtesham Nasreen Z. , Bentur Jagdish S. , Bennett John
Published: May 2005

We describe multicopy DNA clones isolated from a partial genomic library of Orseolia oryzae, based on reverse genomic hybridization, suitable for studying genetic variation in the Asian rice gall midge and other isomorphic species. Three clones produced monomorphic DNA band patterns between biotypes of O. oryzae but polymorphic patterns were produced between O. oryzae and O. fluvialis, the paspalum midge. These probes detect changes in the repetitive sequence structure between species and constitute the first genetic markers for distinguishing between field isolates of rice gall midge and related species of Orseolia. These will be useful in identifying and perhaps eradicating alternative hosts for this pest, and detecting early‐season outbreaks of O. oryzae from light trap collections.
Hypervariable genomic variation to reconstruct the natural history of populations: Lessons from the big cats
Authors: Menotti‐Raymond Marilyn , O'Brien Stephen J.
Published: May 2005

The extent and nature of variation in hypervariable regions of DNA have been used in the past as a means to infer the natural histories of populations. We review the interpretation of the extent of genetic diversity for minisatellite DNA in the cheetah to estimate the timing of a population bottleneck in the species and the potential application of a second class of hypervariable DNA, microsatellite DNA, as a molecular tool to examine the natural histories of felid populations. A calibration curve relating the degree of allele fragment sharing in individuals to relatedness in a captive pedigree of cheetahs is presented. This measurement has important applications for management of potential matings in captive management situations.
Social and related implications of DNA fingerprinting and other new DNA technologies
Authors: Bhargava Pushpa M.
Published: May 2005

The discovery of the structure of DNA at the beginning of this half of the century, and the recognition that it is virtually the universal genetic material, made it imperative for man, sooner or later, to apply this knowledge for unexpected ends. Genetic engineering, DNA fingerprinting, sequencing of whole genomes (be they of man, plants or microorganisms), or exploitation of the differences between the DNA of the male and the female (for example, of the X and the Y sperm) have thus all been historical imperatives. Today, many such predicted technologies are a reality or have come close to being so. Such DNA‐based technologies have enormous social implications, discussed in this article. The impact of these technologies on different societies is likely to be different, in view of the different social conditions that prevail, and the different historical background and cultural traditions. In India, for example, the use of DNA fingerprinting could prevent the large‐scale exploitation of women, and several kinds of litigation. On a global scale, the availability of this technique and its refinement to a stage where it would become virtually foolproof could act as a deterrent to certain kinds of crime. There will also be unprecedented benefits to agriculture but also new problems that will need to be faced and resolved. Wisdom demands that we prepare ourselves for a largescale application of the new DNA technologies that are a historical imperative, understand the problems that are likely to arise, and plan to cope with them before they descend on us in full force.

This is the text of a popular public lecture given by the author during the Third International Conference on DNA Fingerprinting held in Hyderabad, India, in December 1994.
Fluorescence‐based classification of microsatellites using a single‐wavelength semiautomatic sequencer: Genotype assignment and identity tests by analysis of comigrating peak profiles
Authors: Moscetti Alessandra , Boschi Ilaria , Dobosz Marina , Destro‐Bisol Giovanni , Pescarmona Marina , d'Aloja Ernesto , Pascali Vincenzo L.
Published: May 2005

Microsatellite analysis, based on fluorescein labeling and reading through a semiautomatic single wavelength sequencer, is described. Paris of labeled polymerase chain reaction (PCR) samples, mixed in equimolar proportion, were electrophoresed and the specific peaks read in a single gel lane. Identity was asserted when peaks overlapped in a unique fluorescent signal which, compared with individual sample profiles, had a twofold intensity. Classification was achieved by blending individual PCR products to ‘locus specific allelic ladders’ (composite samples containing a repertory of fragments allelic to a given locus) and by noticing the specific peak enhancement. The resulting protocol of analysis assigned no size and classified allelic forms by tandem repeat number. Applied to a large repertory of PCR products and compared with manual electrophoresis, this protocol proved to be reliable and reduced times and costs of genotype analysis. Analysis of comigrating peak profiles is highly objective and provides convincing evidence for diagnostics and identity tests.
Flanking region sequences and internal repeat structure of the pYNH24 (D2S44) 2 kbp insert analyzed by polymerase chain reaction and partial digestion with RsaI
Authors: Holmlund Gunilla , Lindblom Bertil
Published: May 2005

The 2 kbp YNH24 pUC18 insert was partially sequenced, amplified with fluorescence‐labeled primers, and characterized by incomplete digestion with the restriction enzyme RsaI. A characteristic RsaI profile with one restriction site in each repeat unit was obtained when the fragments were analyzed on a DNA sequencer. We developed this procedure with the specific aim of analyzing the internal repeat structure in recombinant alleles often observed in the YNH24 variable number of tandem repeat system (D2S44). Nonetheless, it should also be possible to apply the method when analyzing other tandem repeats, even when little information regarding their sequences is available. In addition, when the repeat array is amplified, either a 5′ or a 3′ fluorescence‐labeled primer can be used to enable analysis from both directions of fairly long (> 3.6 kbp) alleles. The RsaI fragments obtained by partial digestion of the amplified pYNH24 insert corresponded well to the RsaI sites found in the sequenced regions. The 5′ flanking region contained eight 30–34 bp sequences similar to the repeat unit and also several RsaI sites, whereas only a few RsaI sites and no repeat units were found in the 3′ flanking region. Twenty‐five consecutive RsaI sites were revealed, in the center of the pYNH24 insert, indicating a region with strictly repeated core sequences; this repetitive block comprised less than half of the total insert. Two approximately 2.24 kbp HinfI alleles from two different heterozygous individuals were found to have identical internal structure, which was very similar to that of the insert although it exhibited only 15 RsaI repeats.
Theoretical basis of one‐dimensional genome scanning: A direct method to identify the site of a mutation
Authors: Gondo Yoichi , Brilliant Murray H.
Published: May 2005

Genome scanning is a technique designed to uncover a net genetic difference between otherwise identical DNA samples. As such, it can be used to directly identify the site of a gene mutation, facilitating the cloning of DNA fragments from that site. Unlike other conventional positional cloning methods, one‐dimensional genome scanning does not require prior knowledge of the location of the gene or mutation nor does it require closely linked markers. Rather, this method can directly identify the site of a net genomic change, such as a deletion or duplication caused by a mutation. Thus, the genome scanning method can be used in place of classic positional cloning strategies because prior positioning or mapping of the objective gene is unnecessary. By using this approach, we have identified and cloned a DNA fragment duplicated in the pun mutation of the mouse pink‐eyed dilution locus (Brilliant et al., Science 1991, 252, 566–569). However, no other similar attempt using one‐dimensional genome scanning has been reported so far, in spite of the simplicity of the procedure and its success in identifying and ultimately characterizing the pink‐eyed dilution gene of the mouse. The lack of other reports of its success are perhaps not because of the practical difficulties of this method, but may be due to the false presumption that the probability for directly identifying the mutation site using genome scanning is extremely low. The theoretical probability was calculated and is presented here. We calculate that there is about a 50% chance in identifying a 200 kbp net genomic change (due to a mutation) by scanning 10 000 random sites in the mammalian genome. The practical key elements for calculating the probability of success are also discussed.
Capillary electrophoresis for screening of adenylosuccinate lyase deficiency
Authors: Gross Manfred , Gathof Birgit S. , Kölle Pirkko , Gresser Ursula
Published: May 2005

We report a new screening method for adenylosuccinate lyase (ASase) deficiency using capillary electrophoresis (CE). This enzyme defect causes secondary autism and psychomotor retardation in early childhood. In all body fluids of these patients, two succinylpurine metabolites can be found that are normally not detectable: succinyladenosine and succinylaminoimidazole carboxamide (SAICA) riboside. A Beckman P/ACE 2050 capillary electrophoresis system was used with a 47.1 cm capillary, 75 μm ID, and the P/ACE Beckman UV absorbance detector. Untreated urine, injected for 1 s, was separated in a pH 8.63 borate buffer at 20 kV. The two succinylpurines (migration times 13.36 and 13.60 min) were detected at 254 nm only in urine of patients with ASase deficiency but not in control samples.
Impact of electroosmosis on isotachophoresis in open‐tubular fused‐silica capillaries: Analysis of the evolution of a stationary steady‐state zone structure by computer simulation and experimental validation
Authors: Thormann Wolfgang , Caslavska Jitka , Mosher Richard A.
Published: May 2005

A dynamic computer model for simulation of open‐tubular capillary electrophoresis that includes in situ calculation of electroosmosis along the fusedsilica capillary column has been applied to the characterization of an anionic isotachophoretic system in presence of a cathodic electroosmotic flow. For each column segment, electroosmosis is calculated with the use of a wall mobility, the voltage gradient and the degree of dissociation of the silanol surface groups of the capillary wall. Then, the bulk capillary flow is taken to be the average of all of the segment flows and considered to represent a plug flow. This simple approach enables the combined simulation of the temporal behavior of an isotachophoretic zone structure in presence of electroosmosis. For a model anionic isotachophoretic configuration at pH 6, simulation data reveal the asymptotic formation of a stationary steady‐state zone configuration in which electrophoretic and electroosmotic zone displacements are opposite and of equal magnitude. The position of the stationary boundaries are predicted to be dependent on the selected wall pK and mobility values. For two different instruments, qualitative agreement between experimental data and simulation results obtained with a wall pK between 5 and 6 is demonstrated. However, for the two experimental setups, significant differences in electroosmotic pumping (i.e. wall mobility values) are noted.
Capillary electrophoresis of peptides using rectangular and cylindrical geometries: A comparative study
Authors: Cifuentes Alejandro , Poppe Hans
Published: May 2005

A study on the use of rectangular columns in capillary zone electrophoresis (CZE) is presented. The equations controlling the hydrodynamic flow and the plate height in rectangular capillaries are deduced. Several experiments were carried out in order to test the validity of these equations. Also, the possibilities of these columns in terms of thermal dissipation, efficiency, analysis speed and sample capacity were studied and compared with those for cylindrical tubing. The results from both rectangular and cylindrical columns are compared to each other, employing the parameters mentioned above, and also with those predicted by theory. The advantages, i.e. better heat dissipation, higher speed and larger sample capacity, and drawbacks, i.e. high fragility and incompatibility with some capillary electrophoresis instruments, of employing rectangular geometries in CZE are discussed.
An expanded system of restriction landmark genomic scanning (RLGS Ver. 1.8)
Authors: Okazaki Yasushi , Okuizumi Hisato , Sasaki Nobuya , Ohsumi Tomoya , Kuromitsu Junro , Hirota Norifumi , Muramatsu Masami , Hayashizaki Yoshihide
Published: May 2005

The restriction landmark genomic scanning (RLGS) method is a high‐speed genome scanning system which is based on the concept that restriction enzyme sites can be used as landmarks throughout the genome. It employs direct end‐labeling of the genomic DNA digested with a rare‐cutting restriction enzyme, followed by high‐resolutional two‐dimensional electrophoresis. Recently, this system was further developed to lower cost and to simplify the procedure. This paper reviews the RLGS principle and the breakthroughs enabling its further development. Also presented is the precise protocol of the newest version (RLGS Ver. 1.8) that offers cost effectiveness and an expanded production system. Finally, the advantages of this new RLGS method and prospects for its widespread application are discussed.
Theory of capillary electrophoretic separations of DNA‐polymer complexes
Authors: Hubert Sylvain J. , Slater Gary W.
Published: May 2005

Electrophoretic separation of DNA molecules normally requires the use of an anticonvection, sieving polymer matrix such as a gel or an entangled polymer solution. Recently, it has been suggested that free‐solution separation could be achieved in a capillary if an electrically neutral, friction‐generating molecule is attached to the DNA molecules before electrophoresis is carried out. The electrophoretic mobilities are then predicted to be very large and the resulting separation is expected to yield excellent resolution. The size‐dependence of the electrophoretic mobility is attributed to longer DNA molecules pulling the neutral molecule with a larger electric force, thus eluting earlier than shorter DNA molecules. In this article, we focus on the particular case where one attaches an uncharged, flexible polymer to the end of the DNA. Our self‐consistent model takes into account the deformation and the hydrodynamic resistance of the polymer in the flow. We find various regimes, depending on the intensity of the electric field and the length of the polymer. The most favorable conditions for high‐resolution separation of DNA are described.
Application of chemometric experimental designs in capillary electrophoresis: A review
Authors: Altria Kevin D. , Clark Brian J. , Filbey Sharon D. , Kelly Martin A. , Rudd David R.
Published: May 2005

Various chemometric experimental designs have been employed for the optimisation of capillary electrophoresis (CE) methods. Similar designs have been utilised in the assessment of the robustness of CE methods. The designs employed include central composites, fractional factorials, Plackett‐Burman, simplex and overlapping‐resolution mapping. Optimisation studies have largely concentrated on the use of these designs on selection of the optimal electrolyte composition. The robustness testing studies performed have involved the use of screening designs to identify the critical parameters affecting responses such as migration times and resolution. Further designs such as central composites have then been employed to set method limits following robustness studies. It is concluded that the use of experimental designs and statistical data evaluation in conjunction with personal computer‐controlled CE autosamplers and instruments are of great benefit in the optimisation and robustness evaluation of CE methods.
Expression of cDNA clones by coupled in vitro transcription/translation and transfection into COS‐1 cells: Protein mapping in two‐dimensional gels
Authors: Madsen Peder , Gromov Pavel , Celis Julio E.
Published: May 2005

We present two procedures that can be used to map proteins in two‐dimensional gels if the cDNA is at hand. The first procedure, which is illustrated with the expression of cDNAs encoding the fatty acid binding protein 5 (FABP 5), psoriasin and stratifin, makes use of the in vitro transcription/translation assay marketed by Promega. The procedure is simple and allows the mapping of the primary translation product in a very short time. The second method – which faithfully reproduces post‐translational modifications – is based on the expression of cDNAs transfected into COS‐1 cells using a eukaryotic expression plasmid. This procedure is illustrated with the expression of cDNAs encoding Ha‐ras p21 and rab 11, two small GTP‐binding proteins known to undergo complex modifications.
Uses of digital image analysis in electrophoresis
Authors: Horgan Graham W. , Glasbey Chris A.
Published: May 2005

The information in electrophoretic gels can be extracted visually, or by using specialised computer hardware and software which treat the gels as digital images. We discuss issues which arise in applying digital image analysis to electrophoresis, and comment on the range of computer packages available. We also illustrate how image analysis can make gel interpretation easier and more reliable. Methods covered include gel registration, and warping, mathematical morphology and deconvolution to sharpen images.
The use of sodium dodecyl sulphate‐polyacrylamide gel electrophoresis to confirm the presence of salmon (Salmo salar) eggs in an illegal fish bait
Authors: Scobbie Alan E. , Mackie Ian M.
Published: May 2005

Salmon roe was identified as the major component of a suspected illegal fish attractant using sodium dodecyl sulphate‐polyacrylamide gel electrophoresis to analyse protein extracts prepared from various salmonid eggs and a suspected illegal bait.
Identification of myocardial proteins from two‐dimensional gels by peptide mass fingerprinting
Authors: Sutton Chris W. , Pemberton Kay S. , Cottrell John S. , Corbett Joseph M. , Wheeler Colin H. , Dunn Michael J. , Pappin Darryl J.
Published: May 2005

Two‐dimensional gels offer a powerful method for separating complex protein mixtures, but subsequent methods for analysing individual components, such as protein sequencing and Western immunoblotting, are laborious and slow. The identification of proteins can be accelerated by using a combination of protease digestion and matrix assisted laser desorption‐mass spectrometry (MALDI‐MS). The peptide mass spectrum of a protein represents a unique fingerprint determined by the amino acid sequence and the cleavage properties of the protease. Software has been developed so that peptide masses can be used to search a mass‐based peptide database generated from established protein sequence databases. A list of the closest matching proteins is produced to allow identification of the sample. The strategy was applied to 52 protein spots from human myocardial tissue separated by two‐dimensional electrophoresis (2‐DE) gels and analysed blind. Conditions for optimal trypsin digestion of proteins electroblotted onto polyvinylidene difluoride (PVDF) membranes are described. Mass data were generated from both Coomassie Brilliant Blue and sulforhodamine B‐stained proteins, though the former required destaining prior to digestion. Alkylation of cysteine and oxidation of methionine were significant modifications that influenced the successful identification of a protein spot. Examples are presented to illustrate the advantages and disadvantages of this approach.
Detection of picornavirus genomic and template RNA strands by a novel semi‐nested polymerase chain reaction‐technique and agarose gel electrophoresis
Authors: Gow John , Cash Philip , Behan Wilhelmina , McGarry Frances , Simpson Kathleen , Behan Peter
Published: May 2005

Attempts to evaluate the relative levels of enteroviral genomic and template RNA strands in small biopsy tissue samples from patients have yielded ambiguous data, largely due to the limited amount of RNA available. A novel seminested polymerase chain reaction (PCR) technique was developed to enable RNA levels to be examined more accurately. PCR products were visualised by horizontal agarose gel electrophoresis. This technique was demonstrated on linear single‐stranded plasmid DNA; viral RNA isolated from a human rhabdomyosarcoma (RD) cell line persistently infected with a mutated coxsackie B5 virus (piRD) and two cell lines, RD and HEp2 cells, acutely infected with a wild‐type clinical isolate of coxsackie B5 virus.
Sequence dependent migration behavior of double‐stranded DNA in capillary electrophoresis
Authors: Berka Jan , Pariat Yvan F. , Müller Odilo , Hebenbrock Kirstin , Heiger David N. , Foret František , Karger Barry L.
Published: May 2005

Capillary electrophoresis (CE) with a replaceable linear polyacrylamide (LPA) sieving matrix was used to examine sequence‐dependent migration of double‐stranded DNA fragments. It has been found that DNA conformational effects were significant under high electric field separations, especially using high resolution matrices. Compared to linear DNA‐ladder standards, both anomalously slow and rapid DNA fragments were observed, with the degree of anomalous migration depending on the electric field strength, polymer concentration, column temperature, and background electrolyte (denaturants, sodium and magnesium ions, DNA‐intercalating dyes). By selecting a combination of electrophoretic conditions (e.g. 3% T LPA, elevated capillary temperature, lower electric field strength and addition of DNA intercalating dyes), molecular weight dependent separations were closely restored

Presented in part at HPCE '94, San Diego, CA, February, 1994.

.
Analysis of heat and cold shock proteins in Listeria by two‐dimensional electrophoresis
Authors: Phan‐Thanh Luu , Gormon Thierry
Published: May 2005

The proteins induced by heat and cold shock in Listeria monocytogenes (pathogenic for humans) and L. innocua (nonpathogenic) strains were analyzed by two‐dimensional (2‐D) electrophoresis with the help of a computerized 2‐D gel analysis system. Heat (49°C) and cold (4°C) shock repressed roughly half the number of proteins synthesized at normal temperature (25°C) and decreased the level of numerous other proteins. Conversely, the synthesis of a great number of proteins was enhanced and novel proteins appeared upon temperature stress. There were more proteins induced in the L. monocytogenes strain than in the L. innocua strain. Each stress induced a set of specific proteins. There was overlap between these sets of proteins induced by heat and cold shock. Furthermore, a number of heat or cold shock proteins were found to be induced in both Listeria species and by both heat and cold shock in both species. The induction by heat shock was more intense than that by cold shock. The most strongly induced common stress protein of Listeria had a molecular mass of 17.6 kDa and an isoelectric point of 5.1.
Direct determination of amino acids by capillary electrophoresis/electrochemistry using a copper microelectrode and zwitterionic buffers
Authors: Zhou Jianxun , Lunte Susan M.
Published: May 2005

Sixteeen free amino acids were separated by capillary electrophoresis (CE) using zwitterionic buffers of pH 9.8 and detected amperometrically using a copper microelectrode. Low conductivity buffers make possible the use of higher buffer concentrations. This depresses the electroosmotic flow, leading to a better separation of the amino acids compared to that obtained using strong electrolytes. The use of zwitterionic buffers also minimizes the amount of Joule heating in the capillary, leading to more efficient separations. Perhaps most importantly for electrochemical (EC) detection, these buffers reduce the noise due to high separation current at the EC detector. The increased signal‐to‐noise ratio (S/N) results in much better detection limits for amino acids than those reported previously for capillary electrophoresis/electrochemistry (CEEC) with a copper electrode. Detection limits ranged from 10 to 400 nm, and the response was linear over three orders of magnitude for most of the amino acids. The system demonstrated good long‐term stability and reproducibility with a relative standard deviation (RSD) of less than 5% for both the migration time and peak current (n = 20). This method was employed for the determination of amino acids in urine and in brain microdialysate samples.
High performance capillary electrophoresis of casein phosphopeptides containing 2‐5 phosphoseryl residues: Relationship between absolute electrophoretic mobility and peptide charge and size
Authors: Adamson Nicholas J. , Reynolds Eric C.
Published: May 2005

Multiple phosphoseryl‐containing peptides from casein containing the cluster sequence ‐Ser(P)‐Ser(P)‐Ser(P)‐Glu‐Glu‐ stabilize amorphous calcium phosphate at neutral and alkaline pH and have been shown to be anticariogenic in various in vitro, animal and human experiments. In an approach to obtain insight into the structure and function of these peptides, we previously developed a method for their analysis using high‐performance capillary electrophoresis (HPCE). A linear relationship was obtained between absolute electrophoretic mobility and q/M2/3 where q is the net negative charge of the peptide calculated using relevant pKa values and M is the molecular mass. M2/3 is a measure of the surface area of a sphere that has a volume proportional to the M of the peptide and relates to the frictional drag exerted on the peptide during electrophoretic migration. To examine this relationship further, we have analyzed a range of high‐performance liquid chromatography (HPLC)‐purified cluster, di‐ and triphosphorylated peptides from casein enzymic (trypsin, alcalase and pancreatin) digests using HPCE and attempted to relate their absolute electrophoretic mobility to various models incorporating peptide size and charge. Absolute electrophoretic mobility of casein phosphopeptides containing the cluster sequence‐Ser(P)‐Ser(P)‐Ser(P)‐Glu‐Glu‐ was most closely correlated with q/M2/3. Di‐ and triphosphorylated peptide absolute electrophoretic mobility correlated with both q/M2/3 and In(q+1)/n0.43. However, for both the q/M2/3 and In(q+1)/n0.43 relationships with absolute electrophoretic mobility, the di‐ and triphosphorylated peptides formed a separate linear relationship to that of the cluster peptides. From these relationships, a di‐ or triphosphorylated peptide exhibited a greater absolute electrophoretic mobility than a corresponding cluster peptide with the same q/M2/3 or In(q+1)/n0.43 value. This implies that the cluster peptides have a reduced effective net negative charge due to an electrical double layer effect and/or a larger hydrodynamic volume or a more extended structure relative to the di‐ and triphosphorylated peptides which is associated with a greater frictional drag during electrophoretic migration.
Separation of eleven angiotensin II analogs by capillary electrophoresis with a nonionic surfactant in acidic media
Authors: Matsubara Norio , Koezuka Kiyohiro , Terabe Shigeru
Published: May 2005

Eleven angiotension II analogs of same chain length were separated by capillary electrophoresis at pH 2.0 with 200 mM Tween 20. All compounds except one pair of angiotensin II ([Sar1, Gly8]‐ and [Sar1, Val5, Ala8]‐angiotensin II) were baseline‐separated, even in the case of peptides with about the same total charge. The migration order of the angiotensin II analogs were determined by the hydrophobicity of the amino acid as long as the difference between amino acids of two peptides is the conservative change. From the study of pH dependency of the separation of these peptides, it was found that the conditions of a low electroosmotic flow under a low pH is effective for the separation of similar peptides.
A simplified silver diammine method for the staining of nucleic acids in polyacrylamide gels
Authors: Vari Frank , Bell Kevin
Published: May 2005

High sensitivity and low background, which are the significant features of the procedure of Johansson and Skoog (J. Biochem. Biophys. Methods. 1987, 14, (Suppl.) 33) for silver staining of nucleic acids in polyacrylamide gels, have been improved by excluding ethanol from all solutions and increasing the length of washing steps after sensitisation and staining. Including a fixation step before sensitisation increased the contrast of the bands even at high concentration of nucleic acid, producing a shift in the tone of colour from black to grey instead of gold to yellow bands. This technique is superior in terms of sensitivity and background to a number of other silver staining protocols, three which utilise silver diammine and three which utilise silver nitrate ions.
Reproducibility of mobility in gel electrophoresis
Authors: Zakharov Sergey F. , Chang Huang‐Tung , Chrambach Andreas
Published: May 2005

The quantitative exploitation of gel electrophoresis to yield molecular and gel fiber properties rests on the assumption that mobility is characteristic of the macromolecule migrating as a band and is a physical constant for any system defined by pH, ionic strength and temperature. This assumption has not been tested intra‐experimentally in previous literature. With the commercial introduction of automated gel electrophoresis apparatus, the collection of multiple mobility data during a single run without additional expense of labor has made it possible to test the assumption. As a start, we undertook that test for three proteins and their sodium dodecyl sulfate (SDS) derivatives, in agarose and polyacrylamide gel electrophoresis, various field strengths, continuous and discontinuous buffers, as well as intra‐ and interexperimentally. It was found that in agarose gel electrophoresis conducted in a single buffer, the standard deviation of mobility over a wide concentration range ranges intra‐experimentally from 0.2 to 1.3% for two globular proteins and 1.4 to 5.3% for the same proteins derivatized with SDS. Interexperimentally, it was 3% in the single case tested to date. The standard deviation in polyacrylamide appears to be higher, varies in inverse relation to the mobility value, i.e. increases with gel concentration in the range of 11 to 19%T, and varies substantially between the two SDS‐proteins investigated. Mobility in a discontinuous buffer system decreases continuously due to the decreasing leading phase/trailing phase ratio along the migration path. The decrease is sharpest in the “nonrestrictive” stacking gel.
Investigation of wool protein heterogeneity using two‐dimensional electrophoresis with immobilised pH gradients
Authors: Herbert Ben R. , Chapman Anna L. P. , Rankin Douglas A.
Published: May 2005

The heterogeneity of intermediate filament proteins (IFP) from wool has been investigated using two‐dimensional polyacrylamide gel electrophoresis with immobilised pH gradients in the first dimension. The charge heterogeneity has been confirmed with some of the IFP separated as a string of spots with similar molecular weight, but markedly different in isoelectric point (pI). The molecular weight and pI distribution of the string of IFP changed after treatment with alkaline phosphatase, indicating that some of the heterogeneity is caused by phosphorylation.
Adherence of oral microorganisms to human parotid salivary proteins
Authors: Newman Frances , Beeley Josie A. , MacFarlane T. Wallace
Published: May 2005

Bacterial colonisation of oral surfaces by microorganisms may be dependent on their interaction with specific host receptor molecules. Primary oral colonisers are known to remove specific proteins from parotid saliva. The aim of this study was to determine whether these interactions facilitate microbial attachment to a surface and hence identify specific salivary components as putative host receptor molecules. Parotid saliva was resolved by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and then electroblotted onto nitrocellulose membranes. Suspensions of fluorescently labelled microoganisms were incubated with the blots and salivary components with adherent bacteria identified as fluorescent bands under ultraviolet (UV) transillumination. Species of streptococci known to be early colonisers of the clean tooth surface were found to adhere specifically to certain salivary proteins, especially to basic proline‐rich proteins (PRPs). Polymorphic variations in these patterns could form the basis of differences in oral microflora, susceptibility to oral infections and consequent disease.
Separation of 1‐aminopyrene‐3,6,8‐trisulfonate‐labeled asparagine‐linked fetuin glycans by capillary gel electrophoresis
Authors: Guttman András , Chen Fu‐Tai A. , Evangelista Ramon A.
Published: May 2005

Asparagine‐linked glycans of bovine fetuin were separated by capillary gel electrophoresis after enzymatic release (peptide‐N‐glycosidase F) and labeling via reductive amination by a fluorescent dye, 1‐aminopyrene‐3,6‐8‐trisulfonate (APTS). At low separation pH (2.5) only two dominant peaks were observed. Increasing the separation buffer pH to 4.75 resulted in complete separation of two primary doublets and several minor peaks from the fetuin N‐linked glycan pool. Two of the four major peaks were spiked with purified individual standards and were identified as trisialylated triantennary structures with different sialylation linkages. The other two larger peaks were postulated to be tetrasialylated triantennary structures, based on calculations considering their corresponding glucose unit (GU) values. Effects of the electrophoretic separation parameters, such as gel concentration, electric field strength and temperature on the migration behavior of the two major doublets of the fetuin glycan pool were also thoroughly examined. Our data suggest that the capillary gel electrophoresis separation of the multisialylated branched oligosaccharides with different linkage isomers, released from bovine fetuin, is fundamentally based on their degree of sialylation and hydrodynamic volumes.
Effect of cationic buffer additives on the capillary electrophoretic separation of serum transferrin from different species
Authors: Oda Robert P. , Landers James P.
Published: May 2005

The presence of specific transferrin (Tf) glycoforms in human serum has been shown to correlate with certain clinical syndromes. Hence, the ability to separate and quantitatively measure the various forms of human transferrin has become increasingly important. As a means of evaluating the potential for developing a rapid capillary electrophoresis‐based assay for the analysis of carbohydrate‐deficient transferrins (CDTs), the capillary zone electrophoretic (CZE) analysis of Tfs from several species was evaluated using uncoated capillaries and a separation augmented with cationic additives. With bovine Tf, five peaks (representing different sialylated forms) were partially resolved in borate and baseline‐resolved when 1,4‐diaminobutane was added to the buffer. These same conditions were found to be inadequate for the resolution of the sialo‐forms from other species. Some success was achieved using α,γ‐bis‐quaternary ammonium alkanes instead of the 1,4‐diaminobutane and optimizing the pH for each of the species Tfs. Human Tf was found to be resolved in an uncoated capillary equilibrated with a borate buffer containing millimolar concentrations of decamethonium bromide as a buffer additive. Under these conditions, resolution of the various sialoforms from the iron‐saturated Tf was possible and the glycoforms were found to migrate differently than their iron‐depleted counterparts. Despite the resolution achievable under these conditions, the lengthy analysis time is incompatible with the requirements for a clinical CZE‐based assay.
Dynamics of DNA molecules in agarose gel under sinusoidal electric fields
Authors: Starchev Konstantin , Sturm Jean , Weill Gilbert
Published: May 2005

The dynamics of DNA molecules in agarose gel has been studied by measuring the birefringence in sine wave electric fields. It was found that the birefringence is the sum of a direct current (dc) component and several harmonics of the field frequency. A detailed study of these components was performed. It was found that the dynamics of DNA molecules under these conditions can be explained by a W conformation which is not detected under dc electric fields. The variation of the field frequency gives several relaxation modes in the birefringence signal, reflecting the known dynamic behavior of DNA molecules found in the reptation with elongation of the tube length regime.
Sodium chloride in preparative free‐flow cell electrophoresis: Recent developments
Authors: Bauer Johann , Weber Gerhard
Published: May 2005

Three buffer systems for free‐flow electrophoresis have been designed, which proved useful for performing cell electrophoresis in the presence of 50 mM NaCl. Each system consists of one central cell suspension buffer with 50 mM NaCl, two margin buffers, and two electrode buffers. With the aid of a bromophenol blue/maxilon blue accumulation test the various buffers were adjusted to ensure a laminar flow and remain unchanged on their way through an electrophoresis chamber.
Immunoglobulin G galactosylation deficiency determined by isoelectric focusing and lectin affinoblotting in differential diagnosis of rheumatoid arthritis
Authors: Kötz Kerstin , Hänsler Marion , Sauer Heidemarie , Kaltenhäuser Sylke , Häntzschel Holm
Published: May 2005

IgG galactosylation deficiency in patients with rheumatoid arthritis (RA) can be detected by isoelectric focusing (IEF)/lectin affinoblotting. We analyzed IgG glycosylation in patients with early arthritis (n = 50) and healthy controls in order to determine the clinical value of this parameter in differential diagnosis of RA. A significant correlation between the IgG galactosylation defect at disease onset and the diagnosis of RA during the follow‐up was observed. Involvement of other clinical parameters (erythrocyte sedimentation rate, C‐reactive protein, rheumatoid factor) did not improve the predictive value of IgG galactosylation changes.
Phenotyping of esterase D and acid phosphatase by rapid isoeletric focusing on PhastSystem
Authors: Yaojun Guo , Lili Huang , Xuegang Yao , Zhongyi Yu
Published: May 2005

A rapid isoelectric focusing method on PhastSystem was applied to investigate the distribution of esterase D (ESD) and acid phosphatase (ACP) phenotypes in 152 and 112 unrelated healthy donors, respectively, from the Han population in Beijing. Laboratory‐made gels with a pH range of 5.0–7.0 and 5.0–8.0 were used for analysis of ESD and ACP, respectively, with 1 μL sample loading. The running time was 30–40 min. Gene frequencies for ESD were 0.6809 for ESD*1 and 0.3191 for ESD*2, (∑χ2 = 0.8586 and P ≥ 0.50); gene frequencies of ACP were 0.2009 for ACP*A, 0.7991 for ACP*B, (∑χ2 = 1.7892 and P > 0.20), with good agreement between the observed and the expected values.