bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential display approach to quantitation of environmental stimuli on bacterial gene expression (minireview)
Authors: Fislage Rainer
Published: May 2005

The differential display of the mRNA technique for eukaryotes is fruitful in identifying genes with altered transcription rates caused by exogenous or endogenous stimuli. Prokaryotic analogues of the method using arbitrary oligonuleotides may reach a complete statistical genome coverage. Thus a genomewide mass screening for transcriptionally regulated sequences will be possible. However, the primer sets have to be optimized for a given species to result in maximum band yields. Hence the construction of primers requires the calculation of oligonucleotide frequencey distributions from known coding regions to choose sequences with frequent occurrence in the bacterial genome. After completion of many whole genome sequencing projects, differentially regulated cDNA sequences are readily identified by sequence comparisons.
The gel edge electric field gradients in denaturing polyacrylamide gel electrophoresis
Authors: Desruisseaux Claude , Slater Gary W. , Drouin Guy
Published: May 2005

It has previously been shown that zones of higher electric field form close to the loading end of the gel during denaturing polyacrylamide gel electrophoresis. Here we show that the field can reach up to three times its normal mean value a few cm in front of the loading wells when 44.5 mM Tris‐44.5 mM boric acid‐1 mM EDTA is used as the gel buffer. We also demonstrate that this electric field gradient is mostly due to the difference in ion transference numbers at the gel/buffer interface caused by the high viscosity of the urea solution contained in the gel. This field gradient leads to increased band widths and forces us to redefine both the electrophoretic mobility and the mean field intensity. We discuss some methods that can be used to minimize the effects of this gradient.
Do DNA gel electrophoretic mobilities extrapolate to the free‐solution mobility of DNA at zero gel concentration?
Authors: Strutz Kurt , Stellwagen Nancy C.
Published: May 2005

The electrophoresis of small DNA fragments has been measured in dilute agarose and polyacrylamide gels cast and run in Tris‐acetate‐EDTA (TAE) and Tris‐borate‐EDTA (TBE) buffers. Ferguson plots were constructed to extrapolate the mobilities to zero gel concentration and estimate the free solution mobility of DNA. In polyacrylamide gels, in both TAE and TBE buffers, the extrapolated mobilities at zero gel concentration increased gradually with decreasing DNA molecular weight, went through a maximum at ∼ 60 bp, and then decreased again. The increase in the extrapolated mobilities with decreasing molecular weight observed for DNA fragments ⩾ 60 bp can be attributed to transient interactions between the migrating DNA molecules and the polyacrylamide gel fibers. If such interactions are eliminated by extrapolating the mobilities to both zero gel concentration and zero DNA molecular weight, the apparent free solution mobility of DNA is found to be 3.1 × 10−4 cm2V−1s−1 in TAE buffer and 4.2 × 10−4 cm2V−1s−1 in TBE buffer at 20°C, reasonably close to the actual free solution mobilities measured in the same two buffers by capillary electrophoresis (N. C. Stellwagen et al., Biopolymers 1997, 42, 687–703). The significantly larger electrophoretic mobility observed in TBE buffer is most likely due to the formation of nonspecific, highly charged deoxyriboseborate complexes in this buffer medium. For DNA molecules ⩽ 60 bp in size, the decrease in the extrapolated mobilities with decreasing molecular weight parallels the decrease in their free solution mobilities observed by capillary electrophoresis. In agarose gels, the extrapolated mobilities of small DNA molecules at zero gel concentration appear to be independent of molecular weight. The apparent free solution mobilities are found to be (3.0 ± 0.1) × 10−4 cm2V−1s−1 in TAE buffer and (3.2 ± 0.1) × 10−4 cm2V−1s−1 in TBE buffer. The very similar mobilities observed in the two buffer media suggest that the borate ions in TBE buffer primarily form complexes with the galactose residues in the agarose gel fibers, rather than with the migrating DNA molecules, because of mass action effects. The formation of borate‐agarose complexes, increasing the net negative charge of the agarose gel fibers, appears to be responsible for the markedly increased electroendosmotic flow observed in agarose gels cast and run in TBE buffer (N. C. Stellwagen, Electrophoresis 1992, 13, 601–603).
Enhanced hybridization labeling signals in Southern blotted DNAs fractionated with voltage gradient gel electrophoresis
Authors: Barbieri Rainer , Duro Giovanni , Izzo Vincenzo
Published: May 2005

An enhancement of hybridization labeling signals is demonstrated in Southern blotted DNAs, fractionated by voltage gradient gel electrophoresis. This enhancement is due to a reduced thickness of each single nucleic acid band in the gel as a consequence of the gradient effect, corresponding to an increased concentration of DNA per unit area.
Large scale isolation of genes as DNA fragment lengths by continuous elution electrophoresis through an agarose matrix
Authors: Rodriguez Henry , Akman Steven A.
Published: May 2005

A simple procedure for the isolation of genes as DNA fragment lengths is described. By using a commercial continuous elution protein electrophoresis apparatus and incorporating an agarose matrix, preparative scale amounts (300 μg) of DNA can be purified by fragment lengths from a mixture of genomic fragment lengths with high recovery yields. Fractions corresponding to unique fragment length ranges are screened for individual genes by dot‐blot analysis. Using this technique, we have isolated two genes: PGK1, a single copy housekeeping gene; and p53 (exons 3–11), a tumor suppressor gene, whose DNA fragment lengths elute at 4 and 7.5 kbp, respectively, from a single preparative run. As an example of the utility of the technique, we applied it to improving the sensitivity of the ligation‐mediated polymerase chain reaction (LMPCR) — a nucleic acid amplification technique used for the detection and mapping of DNA damage along genes. By eliminating excess nontargeted genomic DNA, the agarose matrix continuous elution electrophoresis (CEE) procedure provided a 24‐fold increase in signal strength attributable to base damage caused by exposing DNA to reactive oxygen species. Genomic DNA fragment length purification by agarose matrix CEE should also prove useful in other research areas requiring gene isolation, such as genomics and molecular biology.
Detection of clonal T cells in cutaneous T cell lymphoma by polymerase chain reaction: Comparison of mutation detection enhancement‐polyacrylamide gel electrophoresis, temperature gradient gel electrophoresis and fragment analysis of sequencing gels
Authors: Scheller Uta , Muche J. Marcus , Sterry Wolfram , Lukowsky Ansgar
Published: May 2005

Cutaneous T cell lymphomas (CTCL) can be differentiated from benign inflammatory dermatoses by the demonstration of clonal T cells in skin biopsy. As a marker of the T cell clonality, the rearrangement of the T cell receptor (TCR) genes is amplified by polymerase chain reaction (PCR) and subsequently analyzed by several electrophoresis techniques. Since the validity of this approach depends substantially on the separating capacity of the applied electrophoresis technique, we investigated in the present study the lower detection limit and the sensitivity of heteroduplex‐loaded polyacrylamide gel electrophoresis on MDE® (mutation detection enhancement) gels (HD‐MDE PAGE), of heteroduplex‐loaded temperature gradient gel electrophoresis (HD‐TGGE) and fragment analysis (FA) on sequencing gels. Genomic DNA from formalin‐fixed, paraffin‐embedded skin biopsies of 53 CTCL specimens and 27 samples of benign dermatoses was analyzed by TCRy PCR followed by electrophoretic separation. Clonality was detected by HD‐MDE PAGE in 22, by HD‐TGGE in 34, and by FA in 33 of the 53 CTCL cases. Additionally, FA revealed an oligoclonal fragment profile in seven CTCL specimens. In the 27 samples from benign dermatoses, HD‐MDE PAGE and HD‐TGGE showed the expected polyclonal pattern in 26, and FA in 25 specimens. HD‐TGGE and FA detected a clonal pattern down to a dilution of 103 monoclonal cells in 106 peripheral blood mononuclear cells (PBMC), while HD‐MDE PAGE revealed a detection limit of 104 monoclonal cells in 106 PBMC. In conclusion, HD‐TGGE and FA possess a higher sensitivity and lower detection limit than HD‐MDE PAGE. Therefore, both former techniques are useful tools for the routine diagnostic procedure. With regard to time and cost, we recommend HD‐TGGE.
Simultaneous genotyping of alcohol dehydrogenase 2 (ADH2) and aldehyde dehydrogenase 2 (ALDH2) loci by amplified product length polymorphism (APLP) analysis
Authors: Aoshima Toshiaki , Umetsu Kazuo , Yuasa Isao , Watanabe Gotaro , Suzuki Tsuneo
Published: May 2005

Genotyping of the alcohol dehydrogenase 2 (ADH2) and aldehyde dehydrogenase 2 (ALDH2) loci is important in alcohol studies. We describe a method for simulataneous genotyping of ADH2 and ALDH2 based on amplified product length polymorphism (APLP) analysis. Two polymerase chain reaction (PCR) fragments for ADH2 (57 bp, 53 bp) and two for ALDH2 (78 bp, 73 bp) are simultaneously amplified. Nine banding patterns reflecting the genotypes of the ADH2 and ALDH2 loci are clearly and unambiguously distinguished. The APLP method seems to be particularly suited for large‐scale population studies of ADH2 and ALDH2 loci because it is simple and rapid.
Population analysis of the collagen type IIα1 3′ variable number of tandem repeat polymorphism by heteroduplex genotyping
Authors: Uitterlinden André G. , Huang Qiuju , Pols Huibert A. P. , van Leeuwen Johannes P. T. M.
Published: May 2005

The AT‐rich variable number of tandem repeat (VNTR) marker at the 3′ end of the collagen type IIα1 (COL2A1) gene has been shown to have a complex structure with extensive sequence variations among repeat units. We analyzed this VNTR polymorphism in a large population of 972 Caucasian individuals with a genotyping procedure involving heteroduplex analysis of PCR products using polyacrylamide gel electrophoresis. Seven size alleles were identified, combining to 19 different homoduplex genotypes (heterozygosity = 0.64). These could be further dissected by analysis of heteroduplexes into 85 different heteroduplex genotypes (heterozygosity = 0.84). By systematically heteroduplexing homozygous and heterozygous individuals in vitro, characteristic heteroduplex doublet bands were generated of known allelic composition. A comparison of these doublets with heteroduplex patterns observed in the population allowed us to identify 29 alleles. The degree of correspondence with a different COL2A1 VNTR genotyping system, based on size separation of single‐strand VNTR alleles [l], was investigated by the analysis of samples typed with both methods, including samples from reference CEPH pedigrees. This revealed improved genetic resolution by the heteroduplex method including discrimination of frequent alleles considered identical by the single‐strand analysis. Our findings demonstrate heteroduplex analysis of the COL2A1 VNTR to be a robust and highly informative genetic marker system. The documented increased genetic variability has important implications in forensic and paternity testing, as well as in linkage and association studies relating genetic variation at this locus to disease endpoints.
DNA cycle sequencing of a common restriction fragment of Staphylococcus aureus bacteriophages by capillary electrophoresis using replaceable linear polyacrylamide
Authors: Klepárník Karel , Berka Jan , Foret František , Doškař Jiři , Kailerová Jana , Rosypal Stanislav , Boček Petr
Published: May 2005

The nucleotide sequence of a part of a 4.9 kbp common restriction fragment isolated from Staphylococcus aureus bacteriophage (bacterial virus) 3A has been determined by capillary electrophoresis (CE). The fast separation of sequencing fragments in linear polyacrylamide solution at a temperature of 55°C allowed the reading of more than 650 bases of sequence in 60 min. The single strand (ss)DNA fragments were prepared by cycle sequencing with fluo‐rescently labeled dideoxy‐terminators on the cloned bacteriophage DNA template. With respect to analysis speed, sequence read‐length, low sample consumption and automation, CE offers a simple, labor‐saving and inexpensive procedure for DNA sequencing. Operating the CE columns at elevated temperature proved to be a rapid procedure capable of extending sequence read‐length. The resulting sequence of the common restriction fragment can be used for the preparation of specific primers and oligonucleotide hybridization probes for identification of Staphylococcus aureus bacteriophages and/or prophages belonging to the bacteriophage species 3A.
Enhanced separation of antidepressant drugs using a polymerized nonionic surfactant as a transient capillary coating
Authors: Harrell Crystal W. , Dey Joykrishna , Shamsi Shahab A. , Foley Joe P. , Warner Isiah M.
Published: May 2005

The separation of seven structurally similar antidepressant drugs (amitriptyline, nortriptyline, imipramine, desipramine, protriptyline, doxepin, and nordoxepin) was achieved in under 15 min using a novel nonionic micelle polymer, poly(n‐undecyl‐α‐D‐glucopyranoside) (PUG) by use of capillary zone electrophoresis (CZE). Systematic studies with varying polymer concentration, pH, and percent organic modifier were conducted in order to find the optimum conditions for baseline separation of the seven tricyclic antidepressants. In addition, equations for capacity factor were used to estimate the extent of what was initially thought to be micelle analyte interaction. A series of calculations show that a modified CZE system (PUG‐CZE) was the actual mode of separation. Thus, our study concluded that PUG functioned in a non‐electrokinetic chromatography mode.
High sensitivity analysis of proteins and peptides by capillary electrophoresis‐tandem mass spectrometry: Recent developments in technology and applications
Authors: Figeys Daniel , Aebersold Ruedi
Published: May 2005

Analytical biochemistry, in particular the analysis of regulatory proteins that control biological systems and pathways, is dependent on methods of ever‐increasing sensitivity. Capillary electrophoresis (CE) has long been recognized as an ultrasensitive analytical technique. In spite of the high sensitivity, CE has not penetrated protein discovery research as a standard analytical method. In this review article we summarize recent technical developments which have significantly enhanced CE as a tool for the analysis of trace amounts of proteins. Specifically, we review recent advances in the development and application of capillary electrophoresis‐mass spectrometry (CE‐MS) and on‐line analyte concentration techniques, and introduce the emerging field of microfluidics as a front end to mass spectrometry (MS).
Database searching using mass spectrometry data
Authors: Yates John R.
Published: May 2005

Large‐scale DNA sequencing is creating a sequence infrastructure of great benefit to protein biochemistry. Concurrent with the application of large‐scale DNA sequencing to whole genome analysis, mass spectrometry has attained the capability to rapidly, and with remarkable sensitivity, determine weights and amino acid sequences of peptides. Computer algorithms have been developed to use the two different types of data generated by mass spectrometers to search sequence databases. When a protein is digested with a site‐specific protease, the molecular weights of the resulting collection of peptides, the mass map or fingerprint, can be determined using mass spectrometry. The molecular weights of the set of peptides derived from the digestion of a protein can then be used to identify the protein. Several different approaches have been developed. Protein identification using peptide mass mapping is an effective technique when studying organisms with completed genomes. A second method is based on the use of data created by tandem mass spectrometers. Tandem mass spectra contain highly specific information in the fragmentation pattern as well as sequence information. This information has been used to search databases of translated protein sequences as well as nucleotide databases such as expressed sequence tag (EST) sequences. The ability to search nucleotide databases is an advantage when analyzing data obtained from organisms whose genomes are not yet completed, but a large amount of expressed gene sequence is available (e.g., human and mouse). Furthermore, a strength of using tandem mass spectra to search databases is the ability to identify proteins present in fairly complex mixtures.
Capillary column chromatography improves sample preparation for mass spectrometric analysis: Complete characterization of human α‐enolase from two‐dimensional gels following in situ proteolytic digestion
Authors: Reid Gavin E. , Rasmussen Richele K. , Dorow Donna S. , Simpson Richard J.
Published: May 2005

Two‐dimensional polyacrylamide gel electrophoresis (2‐DE) in combination with mass spectrometry is an extremely powerful tool for characterizing complex mixtures of proteins. In many cases, the success of this approach relies upon the ability to recover peptides at high concentrations and free of interfering artifacts from in‐gel and/or on‐membrane enzymatic digests. In previous studies, we demonstrated that capillary or microcolumn ( ≤ 350 μm ID) rever‐sed‐phase high performance liquid chromatography (RP‐HPLC) is a powerful microseparation technique for proteins and peptides (Moritz, R. L. and Simpson, R. J., J. Chromatogr. 1992, 599, 119–130). Here we evaluate various capillary column RP‐HPLC/mass spectrometric approaches for identifying and characterizing 2‐DE resolved proteins. For these studies, stable and efficient 0.20 mm and 0.32 mm internal diameter (ID) fused‐silica columns with hydrophilic polyvinylidene difluoride (PVDF) frits were fabricated and slurry packed with 7 μm spherical, 300 Å pore size, C8 bonded phase silica particles. We show that capillary column chromatography is a rapid and efficient desalting/concentrating (ON/OFF) technique for sample cleanup prior to protein identification by peptide‐mass fingerprinting using matrix‐assisted laser desorption ionization (MALDI)‐time‐of‐flight mass spectrometry. While marginally more peptide mass information can be obtained by stepped elution of the peptide mixture with increasing concentrations of organic solvent, best results were obtained by fractionation of the peptide mixture using a linear 60 min gradient. One salient feature of this study was the observation that, in contrast to the stepped elution and gradient approaches, the ionization of peptide T1 (m/z 2402.2 SGETEDTFIADLVV(PeCys)TGQIK) was almost completely suppressed using the ON/OFF approach. Maximal amino acid sequence coverage, a necessary prerequisite for complete characterization of a protein, was accomplished using a capillary column (0.2 mm ID) directly coupled with an electro‐spray ionization (ESI) ion‐trap tandem mass spectrometer. For example, from an in situ tryptic digest of α‐enolase isolated by 2‐DE from the human breast carcinoma cell line MDA‐MB231, 71% of the amino acid sequence was obtained. In addition to identifying two possible N‐terminal acetylated α‐enolase variants, Asn153Asp and Ile152Asp/Asn153Ile, the tandem mass spectrometric data revealed the presence of a number of process‐induced modifications of α‐enolase such as methionine oxidation and cysteine amidoe‐thylation

Part of this work was presented at ICES '97, International Council of Electrophoretic Societies Symposium, held March 23–27, 1997 in Seattle, WA, USA.
Application of free‐flow electrophoresis to the purification of trichosanthin from a crude product of acetone fractional precipitation
Authors: Song Jin‐Fang , Liu Tao , Shen Xin , Wu Gao‐de , Xia Qi‐chang
Published: May 2005

The application of free‐flow electrophoresis (FFE) to the purification of trichosanthin (TCS) from a crude product of acetone fractional precipitation was investigated. An electrophoresis technique, combining field step electrophoresis (FSE) and zone electrophoresis (ZE) to a one‐step procedure, was optimized until a satisfactory purification factor (1.35), high resolution, and purity (>99%) were achieved. Testing several separation buffer systems revealed that a throughput of 14.2 mg/h can be obtained when the very basic TCS (pI 10.1) was dissolved and electrophoresed in a phosphate buffer system of pH 4. The purity of electrophoresed trichosanthin was proved by a variety of analytical methods, such as sodium dodecyl sulfate (SDS)‐gel electrophoresis, capillary isoelectric focusing (CIEF), and sequencing of N‐ and C‐termini. The high purity and large throughput achieved at low cost by using FFE indicates that this method can be employed for TCS purification.
Isolation of peroxisome subpopulations from rat liver by means of immune free‐flow electrophoresis
Authors: Völkl Alfred , Mohr Heribert , Weber Gerhard , Dariush Fahimi H.
Published: May 2005

Immune free‐flow electrophoresis (IFFE) has been applied to the separation of peroxisomes (PO). IFFE is a modification of antigen‐specific electrophoretic cell separation (ASECS), and combines the advantages of electrophoretic separation with the high selectivity of an immune reaction. It differs from the latter in the pH of the electrophoresis buffer, which was shifted from the physiological range (ASECS) to the pI of IgG molecules (pH ∼ 8.0), thus further decreasing the mobility produced by the binding of a specific antibody. This enhances the mobility differences between IgG‐coupled particles and those nondecorated, with resultant improved separation. We have now succeeded in isolating different subpopulations of PO by applying IFFE to heavy, light, and post‐mitochondrial fractions separated by differential centrifugation of a rat liver homogenate. The obtained PO subfractions differed in their composition of matrix and membrane proteins, as revealed by immunoblotting. This indicates that they indeed represent distinct subpopulations of rat hepatic PO.
High performance density gradient electrophoresis of subcellular organelles, protein complexes and proteins
Authors: Tulp Abraham , Verwoerd Desirée , Benham Adam , Jalink Kees , Sier Cees , Neefies Jacques
Published: May 2005

A density gradient electrophoresis (DGE) apparatus (2.2 Ø, 14 cm) was constructed for the rapid separation of milligram quantities of proteins. By using binary buffers according to Bier (Electrophoresis 1993, 14, 1011–1018) proteins were rate‐zonally separated in less than 60 min. Acidic proteins were separated in a pH 8.6, 56 μS/cm buffer, and basic proteins in a pH 5.4, 76 μS/cm buffer. Thus the A (pI 5.15) and B (pI 5.30) forms of β‐lactoglobulin as well as the sialylated glycoforms of apotransferrin were well separated at pH 8.6. The isoforms of myoglobin (pI 6.9 and 7.35, respectively), RNAse A (pI 9.45) and cytochrome c (pI 10.0) and lysozyme (pI 11) were separated at pH 5.4 within 80 min. On a 7 cm DGE column, subcellular organelles derived from HeLa cells were separated in standard electrophoresis buffer (655 μS/cm) for 90 min at 10 mA. Using a new low conductivity buffer (193 μS/cm) 20 min was sufficient to separate late endosomes, lysosomes, endoplasmic reticulum, early endosomes, plasma membrane, clathrin‐coated pits, proteasomes, and clathrincoated vesicles within a single run directly from a postnuclear supernatant.
Transcriptional regulation of mammalian cytochrome c oxidase genes
Authors: Grossman Lawrence I. , Seelan R. Sathiagana , Jaradat Saied A.
Published: May 2005

The cytochrome c oxidase (COX) holoenzyme is a 13‐subunit complex that carries out the terminal step in the electron transport chain. Three of the subunits, which contain the electron transfer function, are coded by mitochondrial DNA and the other ten subunits by nuclear DNA. Since the holoenzyme contains equivalent amounts of each subunit, we and others have examined transcriptional regulation of COX nuclear subunits to explore whether there is a common basis for co‐regulation. Each gene is seen to have a unique pattern of recognition by regulatory factors; although some factors bind to more than one gene, not all COX genes seem to be regulated by the same set of factors. Current information about the COX promoters that have been examined is summarized, and the relation of promoter regulation to coordinate gene expression is discussed.
Bipolar clamping improves the sensitivity of mutation detection by temperature gradient gel electrophoresis
Authors: Gille Christoph , Gille Andreas , Booms Patrick , Robinson Peter N. , Nürnberg Peter
Published: May 2005

Temperature gradient gel electrophoresis (TGGE) is a rapid and sensitive screening method for point mutations and other small DNA alterations. Usually a polymerase chain reaction (PCR)‐product of 150 to 500 bp that has been clamped at one end by a psoralen molecule or a “GC‐clamp” is tested for abnormal melting characteristics by electrophoresis in a temperature gradient. Under optimal conditions, a heterozygous mutation within the fragment is detected through the presence of three additional bands in the TGGE gel, the mutant homoduplex and two heteroduplex bands. However, the ideal pattern of four sharp bands is not always found due to inconsistencies in melting behavior along the sequence of the DNA fragment under study. Some of these fragments show fuzzy bands that may impede or even prevent the detection of a mutation. Here, we describe a method to overcome this problem by utilizing one psoralen clamp at each end of the PCR product. Using TGGE assays established for exons 16, 17, and 18 of the NF1 gene and for exon 14 of the FBN1 gene as examples, we show that bipolar clamping may transform blurred bands into sharp ones and may visualize mutations that could not be detected by conventional single‐sided clamping.
Single‐strand conformation polymorphism (SSCP)‐based mutation scanning approaches to fingerprint sequence variation in ribosomal DNA of ascaridoid nematodes
Authors: Zhu Xing Quan , Gasser Robin B.
Published: May 2005

In this study, we assessed single‐strand conformation polymorphism (SSCP)‐based approaches for their capacity to fingerprint sequence variation in ribosomal DNA (rDNA) of ascaridoid nematodes of veterinary and/or human health significance. The second internal transcribed spacer region (ITS‐2) of rDNA was utilised as the target region because it is known to provide species‐specific markers for this group of parasites. ITS‐2 was amplified by PCR from genomic DNA derived from individual parasites and subjected to analysis. Direct SSCP analysis of amplicons from seven taxa (Toxocara vitulorum, Toxocara cati, Toxocara canis, Toxascaris leonina, Baylisascaris procyonis, Ascaris suum and Parascaris equorum) showed that the single‐strand (ss) ITS‐2 patterns produced allowed their unequivocal identification to species. While no variation in SSCP patterns was detected in the ITS‐2 within four species for which multiple samples were available, the method allowed the direct display of four distinct sequence types of ITS‐2 among individual worms of T. cati. Comparison of SSCP/sequencing with the methods of dideoxy fingerprinting (ddF) and restriction endonuclease fingerprinting (REF) revealed that also ddF allowed the definition of the four sequence types, whereas REF displayed three of four. The findings indicate the usefulness of the SSCP‐based approaches for the identification of ascaridoid nematodes to species, the direct display of sequence variation in rDNA and the detection of population variation. The ability to fingerprint microheterogeneity in ITS‐2 rDNA using such approaches also has implications for studying fundamental aspects relating to mutational change in rDNA.
Diploid Musa acuminata genetic diversity assayed with sequence‐tagged microsatellite sites
Authors: Grapin Agnès , Noyer Jean‐Louis , Carreel Françoise , Dambier Dominique , Baurens Franc‐Christophe , Lanaud Claire , Lagoda Pierre J. L.
Published: May 2005

The sequence‐tagged microsatellite site (STMS) discrimination potential was explored using nine microsatellite primer pairs. STMS polymorphism was assayed by nonradioactive urea‐polyacrylamide gel electrophoresis. Genetic relationships were examined among 59 genotypes of wild or cultivated accessions of diploid Musa acuminata. The organization of the subspecies was confirmed and some clone relationships were clarified.
Preconcentration and separation of antisense oligonucleotides by on‐column isotachophoresis and capillary electrophoresis in polymer‐filled capillaries
Authors: Barmé Iris , Bruin Gerard J. M. , Paulus Aran , Ehrat Markus
Published: May 2005

Small, single‐stranded, chemically modified oligonucleotides, complementary to a specific gene section, commonly referred to as antisense compounds, are being investigated as potential therapeutic drugs. A number of modified oligonucleotides, in particular phosphorothioates, are in clinical development. Shorter fragments are found as metabolic products. Isotachophoresis (ITP) allows the introduction of large, diluted sample plugs into the separation capillary. In this work, ITP and capillary electrophoresis (CE) in polymer solutions were successfully coupled in a single capillary in a commercial instrument to increase sensitivity with UV detection and to shorten the time for sample pretreatment. It was shown that ITP‐CE can be used as a preconcentration and clean‐up method for phosphodiester‐ and phosphorothioate‐containing samples. Up to 3 μL sample could be injected into the capillary without significantly disturbing the separation performance. ITP‐CE of phosphodiesters directly out of salt‐ and protein‐containing samples could be demonstrated. For phosphorothioates in serum samples an additional sample clean‐up was necessary, due to oligonucleotide‐protein binding. An optimized replaceable polymer solution was developed to increase the separation performance for heterogeneous phosphorothioates. A dextran‐based sieving medium showed a good separation performance in ITP‐CE of phosphorothioates. A concentration detection limit of 8.10−9 mol/L for the 20‐mer phosphorothioate ISIS5132, isolated from rat serum, was found.
Identifying human platelet glycoproteins IIb and IIIa by capillary electrophoresis
Authors: Vecchione Gennaro , Margaglione Maurizio , Grandone Elvira , Colaizzo Donatella , Cappucci Giuseppe , Giuliani Nicola , d'Addedda Marina , D'Andrea Giovanna , Nobile Michele , Amoriello Aldo , Minno Giovanni Di
Published: May 2005

Glanzmann thrombasthenia (GT) is an inherited hemorrhagic defect due to a failure of the platelet membrane glycoprotein (GP) IIb–IIIa complex. Capillary electrophoresis (CE) analysis of solubilized platelet membranes from normal individuals showed the presence of two peaks with a migration time of 27 and 29 min, respectively. An excellent run‐to‐run and day‐to‐day reproducibility of the technique (< 1% variation of the retention time) was documented. Using an automated Ferguson method, the apparent molecular masses were 100.0 kDa and 138.5 kDa, respectively. Immunoprecipitation with monoclonal antibodies anti‐GP IIIa (B59.2.1) and anti‐IIb (61.9.1.3) showed the two peaks as IIIa and IIb, respectively. Electropherograms of a GT young man showed the lack of both peaks. Less than 50% of each peak was present in his parents. Polyacrylamide gel electrophoresis (PAGE), immunoblotting, and flow cytometry analyses showed that GP IIb and IIIa were undetectable in the platelet membranes from the propositus, half of the normal amount being present in both parents. These findings indicate CE to be a rapid, sensitive and reliable tool to investigate patients with abnormalities of the GP IIb–IIIa complex.
Capillary electrophoresis for simultaneous quantification of human proinsulin, insulin and intermediate forms
Authors: Arcelloni Cinzia , Falqui Luca , Martinenghi Sabina , Pontiroli Antonio E. , Paroni Rita
Published: May 2005

Capillary electrophoresis (CE) for the simultaneous and precise quantification of human insulin (hI), proinsulin (hPI) and intermediate forms (des 31, 32; split 65–66 and des 64, 65), released in culture media by engineered cells, is described. Analytical conditions for standard proteins were optimized using a bare silica capillary (20 cm × 50 μm internal diameter). Proteins were monitored at 200 nm and separated at constant voltage. Culture supernatants (12–24 mL) were purified on Sep‐Pak Vac C18 cartridges, recovered in 1 mL of acetonitrile:trifluoracetic acid mixture (60:40, v:v), concentrated, ultrafiltered and injected into CE. Protein recovery was 85 ± 14% (n = 5, mean ± standard deviation) with a sensitivity limit of 0.5 nmol/L in the culture media, corresponding to 2 fmol injected in 22 nL. Using the CE method, it was possible to detect and quantify, with precision and accuracy, the release of hPI, hI and intermediate forms directly in the cell culture media, and to compare the proteic pattern released from engineered cells transduced with different hPI gene constructs.
Migration of single‐stranded DNA in polyacrylamide gels during electrophoresis
Authors: Pluen Alain , Tinland Bernard , Sturm Jean , Weill Gilbert
Published: May 2005

By using fluorescence recovery after photobleaching (FRAP) and electric birefringence, the migration of single‐stranded DNA in polyacrylamide gels and orientation as a response to an electric pulse were investigated. Electrophoretic mobility is in good agreement with the model of biased reptation including fluctuations. The determination of the electrophoretic mobility in solution, μo, allows an estimation of the gel pore diameter seen by the molecule. As previously observed for double‐stranded DNA, the electric birefringence results from two processes: the alignment of the molecule along the electric field and the elongation of the primitive path in the gel, for long single‐stranded DNA (> 2000 bases). The combination of results obtained with the two techniques allows us to propose experimental conditions to improve the separation of single‐stranded DNA with pulsed field techniques.
A nonradioactive method for sizing most D11S533 alleles discretely
Authors: Gaudin Daniela , Schmidtke Jörg
Published: May 2005

A method is described which allows accurate nonradioactive sizing of most D11S533 alleles, which are characterized by a highly variable number of 6 bp units. In a small population sample of German origin the heterozygosity of the locus was estimated to be 0.933.
Discontinuities of pH at zone boundaries in isotachophoretic systems with poorly buffering leading electrolytes
Authors: Reijenga Jetse , Kašička Václav
Published: May 2005

Discontinuities of pH at zone boundaries of strong ion sample zones in isotachophoretic systems with poorly buffering leading electrolytes have been discovered by dynamic computer simulations of isotachophoretic separations with uncommon electrolyte systems. When using a salt solution containing a strong cation (anion) as leading ion and a weak anion (cation) as counterion for the isotachophoretic separation of strong cations (anions), severe pH discontinuities are present at zone boundaries of these cations (anions). The magnitude of these pH discontinuities has been investigated as a function of several parameters, namely counter ion/leading ion concentration ratio of the leading electrolyte, pK values, and mobilities of sample ions and counterion.
The band areas of proteins determined by fluorescent scanning in the commercial automated gel electrophoresis apparatus
Authors: Zakharov Sergey F. , Kwok Steven H. , Sokoloff Helen , Chang Huan‐Tsung , Radko Sergey P. , Chrambach Andreas
Published: May 2005

An automated gel electrophoresis apparatus, recently available commercially, allows one to follow the band during electrophoresis in real time, and lends itself therefore to an evaluation of bandwidth as a function of migration time (the dispersion coefficient), resolution and band shape. These determinations assume the constancy of band area with migration time and at various gel concentrations. The purpose of the present study was to verify these assumptions. Representative proteins and sodium dodecyl sulfate (SDS)‐proteins, either natively fluorescent or fluorescein carboxylate labeled, were found to exhibit band areas which approach constancy as a function of migration time in both agarose and polyacrylamide gel electrophoresis, provided that (i) the protein concentration under the band was low enough to obviate self‐quenching of fluorescence; (ii) the separation of the protein of interest from contaminants had progressed sufficiently during the time at which band areas were measured; (iii) the baseline under the peak was sufficiently well defined. However, band areas decrease with increasing gel concentration. Protein peaks exhibited leading and trailing tails. The ratio of the combined tail area to total area appeared to be near‐constant at varying migration times. However, that ratio increases with increasing gel concentration. The tail area does not appear to be an artifact of fluorometric detection since it is reproduced upon fluorimetric analysis of the protein eluted from gel slices after electrophoresis. However, it may be due to photochemical destruction under the conditions of repetitive fluorometric peak detection.
Immunoblotting of multiple antigenic peptides
Authors: Kurien Biji T. , Jackson Kenneth , Scofield R. Hal
Published: May 2005

Multiple antigenic peptides (MAPs) can be efficiently separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to a nitrocellulose membrane for subsequent use in immunoblot (IgG and IgM). MAPs can be stained by Coomassie and silver on SDS‐PAGE as well as by Fast Green on an immunoblot. Affinity immunoblotting for analysis of antibody clonotype distribution has also been carried out using MAPs. High performance liquid chromatography purification of the MAPs is mainly responsible for their migration as sharp bands in SDS‐PAGE and immunoblotting.
Sensitivity models in simulation of capillary zone electrophoresis: The effect of buffer power and initial buffer concentration
Authors: Blanco Stéphane , Clifton Michael J. , Joly Jean‐Louis
Published: May 2005

Separation of proteins by capillary zone electrophoresis in an amphoteric medium is investigated by numerical simulation. We explain how sensitivity models can be used to gain further information about this process. The sensitivity models that are derived are used to study the sensitivity of the separation to the specific buffer capacity of the amphoteric molecule that serves as a buffer. The effect of changing the overall concentration of this ampholyte is also examined and it is shown that a change in its concentration is equivalent to a change in its buffer capacity.
Response patterns with indirect UV detection in capillary zone electrophoresis
Authors: Lu Bing , Westerlund Douglas
Published: May 2005

Capillary zone electrophoresis with indirect UV‐detection was used to separate mixtures containing both positively and negatively charged species. In order to understand the dependence of detector response patterns on the changes in compositions of the background electrolytes and the charge of marker ions (UV‐absorbing ions), the separations were performed in two different systems. In a three‐ion system (analyte ion, coion and counterion) a marker ion was the major ionic component of a buffer solution and in a two‐coion or counterion system the marker ion was used as an additive. In the three‐ion system the response profile of an analyte was in good agreement with the mathematical treatment based on the Kohlrausch regulation function. In the two‐coion or counterion system the response patterns were more complicated; however, the experimental results agree well with data obtained from a computer simulation program. Peak directions of the analytes were not only determined by their relative charge to the marker ion, but were also associated with their relative mobilities to the buffer coion and the marker ion. The analytes with higher effective mobilities compared to the marker ion were detected as positive peaks and the ones with lower effectice mobilities as negative peaks. Similarly to the three‐ion system, the detector response of an analyte was stronger by applying a marker coion compared to a counterion. An interesting result was obtained in the separation of a mixture of quaternary ammonium ions and sugars by using a cationic marker ion. The highest and most symmetrical peak was not a cation, but raffinose anion, which appeared most closely to the system peak. The observation suggests that the electromigration dispersion in its zone was eliminated by migrating close to the electroosmosis. A system peak with the mobility corresponding to the electroosmotic flow was obtained in both systems, and an additional system peak with a mobility close to that the marker ion was present in the systems using marker ions as additives.
High resolution capillary electrophoretic separation of oligonucleotides in low‐viscosity, hydrophobically endcapped polyethylene oxide with cubic order
Authors: Magnusdottir Soffia , Viovy Jean‐Louis , François Jeanne
Published: May 2005

A triblock self‐associating polymer with the structure n‐ dodecane‐poly(ethylene oxide)‐n‐dodecane and a very low polydispersity has been used as a matrix to separate a sample of single‐stranded oligonucleotides containing Pd(A)25–30 and Pd(A)40–60. Above a concentration of 4%, this associative polymer forms a micellar network with cubic order and a well‐defined micellar spacing, in which the dodecane micellar cores are bridged by polyoxyethylene segments. This medium combines a low viscosity with excellent resolution of oligonucleotides. This work confirms that associative polymers are potentially powerful media for separation in capillary electrophoresis, and argues in favor of the use of monodisperse products presenting a high‐order in the physical gel state.
Capillary zone electrophoresis of ds‐DNA in isoelectric buffers: Effect of adding of competing, nonamphoteric ions
Authors: Gelfi Cecilia , Mauri Daniela , Perduca Massimiliano , Stellwagen Nancy C. , Righettis Pier Giorgio
Published: May 2005

When separating ds‐DNA in isoelectric His buffer (pHpI7.6), in the 50–250 mM concentration range, some unique phenomena were observed: improved resolution for smaller DNA fragments, up to ca. 150 bp, and a rapid deterioration of resolution above this critical length (which corresponds to the persistence length). Such phenomenon depended also on voltage and concentration of sieving liquid polymer. Direct binding of His to the DNA helix was hypothesized, with resultant stiffening and an increment of diameter of the DNA fragments, thus inducing an early onset of reptation at the applied voltage in the 100–300 V/cm range. In order to prove this hypothesis, “competing ions” (notably NaCl and KBr) were added to the His background electrolyte: a partial reversal of the His effect was already apparent at low concentrations of such ions (10 mM) and was complete at higher concentrations (30 and 50 mM). By molecular modeling, it was found that His could be docking on the negatively charged oxygen (bound to the phosphate) by offering both charged (primary and tertiary amino) groups to simultaneous binding, thus forming a salt and neutralizing the negative charge borne by the oxygen. The following characteristic bond distances were found: 0.34 nm between the N (imidazolic) and O; 0.32 nm between the primary N and O; 0.36 nm between the two nitrogens engaged in salt formation with the oxygen. In addition, for complexation to occur, the distance between the noncharged nitrogen in the imidazole ring and the nearest phosphate oxygen (engaged in the phosphodiester bridge) should be 0.44 nm. Under these conditions, the two rings present (a six‐membered, ideal one, salt‐linked with the oxygen and rather highly elongated, and the imidazole) will not be precisely coplanar, since the primary and tertiary nitrogens will be one slightly above and one slightly below the plane of the drawing. Upon extensive binding, occupying every available phosphate site, π‐π interactions could occur among the stacks of bound His residues, thus further stabilizing the complex.
On‐line partial filling micellar electrokinetic capillary chromatography‐electrospray ionization‐mass spectrometry of corticosteroids
Authors: Wiedmer Susanne K. , Jussila Matti , Riekkola Marja‐Liisa
Published: May 2005

The one‐line combination of micellar electrokinetic chromatography (MECC) with electrospray ionization‐mass spectrometry (ESI‐MS) was investigated using some corticosteroids as model components. The on‐line technique is difficult because the micelles in the electrolyte solution tend to soil the mass spectrometer and lower the sensitivity of the spectrometer. To prevent the micelles from reaching the mass spectrometer, several techniques have been developed among which is the partial filling (PF) technique. In this study the PF‐MECC technique was investigated in an on‐line MECC‐ESI‐MS study of mixtures of corticosteroids. Because the compounds are uncharged, partitioning or interaction with micelles is required to achieve separation. Surfactant solutions of sodium dodecyl sulfate (SDS), sodium cholate (SC), and SDS/SC mixtures were used as micellar phase. Good MECC separation was achieved after optimization of the surfactant concentrations and the length of the injected micellar plug. Both hydrodynamic and electrokinetic injections of the micellar solutions were tested. A basic ammonium acetate solution was used as the CE electrolyte solution. The ESI‐MS analysis of the compounds was performed in the positive ionization mode, using an acidic sheath liquid. Because of the low MS intensities of the corticosteroids, the peaks were isolated during the MS runs. MS‐MS and MS‐MS‐MS data on the corticosteroids were obtained by off‐line injection of the compounds.
Rapid estimation of octanol‐water partition coefficients of pesticides by micellar electrokinetic chromatography
Authors: Wu Yuan Sheng , Lee Hian Kee , Li Sam F. Y.
Published: May 2005

Micellar electrokinetic chromatography (MEKC) was evaluated as a new technique for the rapid estimation of octanol‐water partition coefficient (logKow). Retention measurements for more than 40 reference pesticides with varied structural characteristics and hydrophobicity were carried out in two MEKC systems, based on sodium dodecyl sulfate (SDS) and sodium cholate (SC), respectively. To enable an accurate determination of capacity factors in the SC‐MEKC system, cypermethrin (a synthetic pyrethroid insecticide) was utilized instead of Sudan III as the SC micelle tracer, since a few highly hydrophobic pesticides were found to elute after Sudan III. The linear correlation between logarithmic capacity factor (logk̃′1) and logKow in the two systems was examined. It was found that, under the typical buffer condition (10 mM sodium phosphate with 60 mM surfactant, pH 7.0), the SDS‐MEKC system provided a somewhat wider dynamic range for hydrophobicity (logKow from −1.0 to 4.5). However, the correlation of logk̃′i with logKow was not very high when all the reference pesticides were included in one single calibration set. For the SC‐MEKC system, the dynamic range for logKow was in the range of 1.0–5.5, and a good linear correlation existed between logk̃′i and logKow, even when all reference pesticides were incorporated into a single calibration group. By comparing the regression line of the reference pesticides with that of a group of simple aromatic derivatives, it was discovered that molecular size and functionality posed a less significant effect on the measurement of logKow in the SC‐MEKC system than in the SDS‐MEKC system. Thus, SC‐MEKC shall be the system of choice for the estimation of logKow. The typical error on logKow determination using the current MEKC technique was within 0.5 units, suggesting that MEKC can be a valuable complement to reversed phase – high performance liquid chromatography (RP‐HPLC) for the indirect determination of logKow. Besides maintaining all the advantages of the HPLC approach, the MEKC technique showed some unique benefits, such as better inter‐column reproducibility, higher throughput, and less handling of toxic pesticides and solvents.