bluuebunny/crossref_metadata_2025 huggingface dataset:
One‐dimensional genome scanning: Identification of the basis of a mouse mutation and identification of genomic changes in ovarian carcinoma
Authors: Brilliant Murray H. , Gondo Yoichi , Magliocco Anthony
Published: May 2005

We have developed a simple one‐dimensional electrophoretic method, genome scanning, that can be used to identify large‐scale genomic differences between two or more DNA samples. Genome scanning is especially useful in the detection of genetic amplifications, deletions, and rearrangements. The assay is essentially a high‐resolution Southern analysis, comparing equivalent amounts of genomic DNA samples that are variant for a given trait. The Southern blots are hybridized to a probe sequence derived from a medium copy number repetitive element (1000–2000 copies per haploid genome) naturally dispersed throughout the genome. The hybridization pattern that results is complex and consists of hundreds of bands. If the DNA samples are otherwise equivalent, a net difference in hybridization intensity between homologous bands of different samples indicates a genetic change. In this report, we discuss the origin of the method, its premise, and review its application to mouse mutational analysis and to human cancer research (a more detailed discussion of the theory is presented elsewhere in this issue; Y. Gondo and M. H. Brilliant, Electrophoresis 1995, 16, 174–178).
The potential of microsatellites for hybridization‐ and polymerase chain reaction‐based DNA fingerprinting of chickpea (Cicer arietinum L.) and related species
Authors: Sharma Prakash C. , Hüttel Bruno , Winter Peter , Kahl Günter , Gardner Richard C. , Weising Kurt
Published: May 2005

The genetic variability in agronomically important chickpea accessions (Cicer arietinum L.) as detected by single‐locus restriction fragment length polymorphism (RFLP) probes, random amplified polymorphic DNA (RAPD) and isoenzyme markers, is rather low. Recently, highly polymorphic microsatellites became the markers of choice for linkage mapping and population studies. We are currently following two main strategies to exploit the variability of microsatellites and adjacent sequences for genetic studies in chickpea. (i) In an approach referred to as oligonucleotide fingerprinting, microsatellite‐complementary oligonucleotides were employed as multilocus probes for in‐gel hybridization. A total of 38 different probes representing di‐, tri‐ and tetranucleotide repeats were used to analyze variability between and within four accessions of C. arietinum. Hybridization signals were obtained with 35 probes. While the abundance and level of polymorphism of the different target sequences varied considerably, distinct, intraspecifically informative banding patterns were obtained with the majority of probes and all restriction enzymes tested. No obvious correlation existed between abundance, fingerprint quality, and sequence characteristics of a particular motif. (ii) In a recently developed strategy called microsatellite primed polymerase chain reaction (MP‐PCR), micro‐satellite‐complementary oligonucleotides serve as single PCR primers for genomic DNA templates. We tested the general applicability of MP‐PCR by amplifying DNA samples from tomato, chickpea and two related annual Cicer species with a variety of di‐, tri‐ and tetranucleotide repeat primers. Most but not all primers generated distinct fingerprint‐like banding patterns after agarose gel electrophoresis and ethidium bromide staining of the amplification products. Since the method proved to be sensitive to reaction conditions in a way similar to RAPD analysis, we increased the PCR specificity by the introduction of a modified “touch‐down” protocol. In chickpea, touch‐down MP‐PCR generated highly reproducible banding patterns which predominantly revealed interspecific polymorphisms. The potential of different microsatellite‐based strategies for genome analysis in chickpea is discussed.
DNA fingerprinting in captive population of the endangered Siberian crane (Grus leucogeranus)
Authors: Tokarskaya Olga N. , Petrosyan Varos G. , Kashentseva Tatiana , Panchenko Vladimir G. , Ryskov Aleksei P.
Published: May 2005

DNA fingerprinting was used to estimate genetic diversity within the endangered Siberian crane (Grus leucogeranus) captive population consisting of several dozens of founders originating from the two wild populations of eastern and western Siberia. Similarity and difference among captive individuals were demonstrated by the unweighted pair‐group (UPGMA) clustering procedure. Quantitative characteristics of the eastern and western captive population groups such as average percentage differences (APD) and heterozygosity showed a high extent of genetic variability of 77.9–79.3% and heterozygosity of 0.85–0.72 within each group. Genetic heterogeneity of the captive population structure observed here provides guidelines for management of the species gene pool in captivity. These data also indicate that monitoring of genetic diversity through DNA fingerprinting can facilitate the efforts of Siberian crane management and restoration.
Size‐selected genomic libraries: The distribution and size‐fractionation of restricted genomic DNA fragments by gel electrophoresis
Authors: Gondo Yoichi
Published: May 2005

By using one‐dimensional genome scanning, it is possible to directly identify the restricted genomic DNA fragment that reflects the site of genetic change. The subsequent strategies to obtain the molecular clones of the corresponding restriction fragment are usually as follows: (i) the restriction of a mass quantity of an appropriate genomic DNA, (ii) the size‐fractionation of the restricted DNA on a preparative electrophoresis gel in order to enrich the corresponding restriction fragment, (iii) the construction of the size‐selected libraries from the fractionated genomic DNA, and (iv) the screening of the library to obtain an objective clone which is identified on the analytical genome scanning gel. A knowledge of the size distribution pattern of restriction fragments of the genomic DNA makes it possible to calculate the heterogeneity or complexity of the restriction fragment in each size‐fraction. This manuscript first describes the distribution of the restriction fragments with respect to their length. Some examples of the practical application of this theory to genome scanning is then discussed using presumptive genome scanning gels. The way to calculate such DNA complexities in the prepared size‐fractionated samples is also demonstrated. Such information should greatly facilitate the design of experimental strategies for the cloning of a certain size of genomic DNA after digestion with restriction enzyme(s) as is the case with genome scanning.
A discontinuous buffer system increasing resolution and reproducibility in DNA sequencing on high voltage horizontal ultrathin‐layer electrophoresis
Authors: Carninci Piero , Volpatti Federico , Schneider Claudio
Published: May 2005

A novel discontinuous buffer system for DNA sequencing based on horizontal ultrathin‐layer gel electrophoresis is described. The optimized system, named unbuffered stacking gel/discontinuous borate EDTA‐buffer system, is composed of a 0.5 mm thick stacking gel, where standard sequencing reactions (1 μL volume) are easily loaded, and a 50 μm ultrathin running gel, where DNA fragments are separated. The novel discontinuous buffer system allows for sample concentration and efficient injection from the stacking gel into the capillary slab gel. Increased resolution, assessed by autoradiography, can be achieved within 25 min running time already over a 10.1 cm distance from the gel slot compared to the conventional gel system. An advantage of the new system is the capacity to resolve compressions in GC‐rich regions, usually causing migrating artifacts in standard gels. The described system affords a major improvement in speed, resolution and reproducibility in DNA sequencing.
Three‐dimensional imaging of DNA fragments during electrophoresis using a confocal detector
Authors: Brewer Laurence R. , Davidson J. Courtney , Balch Joseph W. , Carrano Anthony V.
Published: May 2005

We have measured the three‐dimensional distribution of DNA fragments within an electrophoretic band confined by a glass microchannel. The measurements were made using a confocal microscope and a photon‐counting photo‐multiplier detector. A DNA sequencing standard was loaded into glass micro‐channel plates containing polyacrylamide gel. The measurements were made by scanning the plates in three dimensions using a mechanical stage under computer control, while electrophoresis was taking place. We found that the distribution of DNA was the same for all the bands measured in the sequencing ladder with an approximate Gaussian distribution along all three axes. These measurements are important to understand what physical forces shape electrophoretic bands confined by a channel and they also aid in the design of high throughput DNA sequencers.
Preparative separation of nucleosome core particles containing defined‐sequence DNA in multiple translational phases
Authors: Harp Joel M. , Palmer Elise L. , York Melissa H. , Gewiess Andreas , Davis Matthew , Bunick Gerard J.
Published: May 2005

The nucleosome core particle is composed of an octamer of core histone proteins and about 146 bp of DNA. When reconstituted from purified histone octamer and defined‐sequence, nucleosome positioning DNA fragments, the DNA will bind to the histone core in a number of translational phases with respect to the dyad symmetry axis of the histone octamer. Only one of these phases contains symmetrically bound DNA, and it is this species which is required for crystallization and X‐ray diffraction studies. We have developed a technique for separating nucleosome core particles, containing defined‐sequence 146 bp DNA, which differ only in translational phasing of the DNA with respect to the histone octamer core.
A simple and rapid technique for postelectrophoretic detection of proteases using azocasein
Authors: Peyronel Daniel Vázquez , Cantera Ana María B.
Published: May 2005

Azocasein, diffused into gels after polyacrylamide gel electrophoresis, provides a useful substrate for simple and rapid protease detection. After electrophoresis, the gels are incubated in an azocasein solution, immersed in trichloroacetic acid and then treated with NaOH. Proteolytic zones appear as uncolored bands on an orange background. The gels can be stored in trichloroacetic acid for months. This technique gives good results with serine proteases, acid proteases and metalloproteases. The method can be used under nondenaturing conditions and in the presence of sodium dodecyl sulfate. The sensitivity of this method is at the submicrogram protein level for trypsin.
Feasibility study of a drug immunoassay based on micellar electrokinetic capillary chromatography with laser induced fluorescence detection: Determination of theophylline in serum
Authors: Steinmann Lone , Caslavska Jitka , Thormann Wolfgang
Published: May 2005

This paper presents principle and first results of a novel competitive binding immunoassay for monitoring of theophylline in human serum. The assay is based upon short time incubation of a mixture of antiserum, containing the antibody raised against theophylline, fluorescein labelled theophylline (tracer) and serum prior to injection of a few nanoliter of this mixture onto a fusedsilica capillary for subsequent separation and analysis of free tracer and the antibody‐tracer‐complex by micellar electrokinetic capillary chromatography with laser induced fluorescence detection. Quantitation based upon multi‐level calibration using the height of the peak produced by the free tracer is shown to provide theophylline serum levels which are in agreement with those obtained by a commercial fluorescence polarization immunoassay and with those determined by micellar elektrokinetic capillary chromatography with direct serum injection and on‐column UV absorption detection.
Multilocus genomic mapping with intracisternal A‐particle proviral oligonucleotide probes hybridized to mouse DNA in dried agarose gels
Authors: Lueders Kira K.
Published: May 2005

Recently, oligonucleotide probes that detect intracisternal A‐particle (IAP) gene subfamilies with a limited number of proviral copies have been shown to be useful multilocus markers. A procedure for hybridization of these probes has been developed and is described. In summary, the main features of the method are the following: (i) A pulse controller is used during agarose gel electrophoresis to improve resolution of restriction fragments in genomic DNA. (ii) Hybridization is performed in a dried gel. (iii) The hybridized gel is washed in tetramethylammonium chloride to eliminate differences in oligonucleotide composition on hybrid stability. Use of the prodecure is demonstrated by genomic mapping of IAP loci in the AXB BXA recombinant inbred mouse strains, identification of hypomethylated loci in tumor cells, and detection of a transposed IAP provirus previously identified as the basis for a mutation at the agouti locus.
Theory of zone separation in isotachophoresis: A diffusional approach
Authors: Gebauer Petr , Boček Petr
Published: May 2005

The qualitative characteristics of isotachophoresis differ substantially from “classical” separation methods such as chromatography or zone electrophoresis. Self‐sharpening zone boundaries and step‐like concentration profiles are the most specific features of this method, which does not allow the description of the isotachophoretic separation in usual “chromatographic” terms, such as resolution or number of theoretical plates. A theory is presented in this paper, combining the usual isotachophoretic separation characteristics with the theory of isotachophoretic zone boundary, which is the only element of the isotachophoretic system with dispersional properties. This allows us to consider situations near the limits of the isotachophoretic method as far as both selectivity and sample amount (i.e., zone size) are concerned. Based on a simplified expression of the concentration profiles across the isotachophoretic zone boundary, separation and separation limits are described and discussed in terms of resolution, selectivity and zone capacity. Equations are derived showing the relationships between resolution, boundary width, selectivity, sample amount, and column and electrolyte conditions. A simple phenomenological equation is presented, expressing isotachophoretic resolution as a function of only sample amount (or sample zone length) and boundary width. A thermo‐dynamic form of this equation is derived, which is shown to be similar to such an expression for resolution in zone electrophoresis. In both cases resolution is a function of sample selectivity, electric field strength and column length. A simple theoretical model for zone capacity is presented, making it possible to estimate the separation performance of isotachophoretic systems. Based on the presented theory, parallels between isotachophoresis and zone electrophoresis are discussed and both methods are compared.
Gene and genome scanning by two‐dimensional DNA typing
Authors: Uitterlinden André G.
Published: May 2005

A major effort in the analysis of DNA currently focuses on identifying genes and their pathological variants underlying disease. Once such disease genes have been isolated a major task of molecular medicine is to identify the spectrum of DNA sequence variations responsible for the aberrant function of such genes. These efforts, however, are hindered by the vast amount of genetic information to scan for variations and the limited capacity of analytical techniques in terms of accuracy and speed. Recently, a number of techniques were developed, so‐called “genome scanning” techniques, which allow complete genomes to be analyzed for sequence variation in parallel, i.e., at multiple sites or loci simultaneously rather than serially at predefined loci. Here we present the background and applications of a particular electrophoretic parallel processing approach, generically termed two‐dimensional DNA typing. The approach is based on separating DNA fragments by two‐dimensional electrophoresis [1], including denaturing gradient gel electrophoresis, thus allowing hundreds of fragments to be simultaneously assessed by comparative analysis for variations in size and sequence. The method is suitable for hybridization analysis with locus‐specific and multilocus probes of genomic DNA restriction fragments derived from human and other DNA, and for analysis of polymerase chain reaction (PCR) fragments derived from large genes. Two‐dimensional DNA typing has been applied, e.g., in linkage analysis of pedigrees, analysis of tumor genomes for rearrangements, and to scan the cystic fibrosis transmembrane regulator gene for sequence variations such as point mutations.
Model of isotachophoresis (displacement electrophoresis) in tapered capillaries
Authors: Šlais Karel
Published: May 2005

The principle and theoretical description of boundary focusing under electrophoretic migration in a tapered capillary are presented. The model describes the gradual volume decrease of a moving boundary between weak electrolytes or ampholytes. The relations between the departure from the local steady state, the ratio of inlet to outlet capillary cross section, the effective mobilities and the selectivity are formulated. They allow the selection of operating conditions such that the departure from the local steady state can be decreased to an acceptable level. The relations predict that isotachophoresis (displacement electrophoresis) in a capillary with a shallow taper can maintain a constant ratio of actual boundary width to local steady state width, provided that the local capillary cross section decreases linearly with length. The significance of some assumptions needed for the model formulation is analyzed. Numerical examples are given indicating the method feasibility.
Dynamic control to improve the separation performance in capillary electrophoresis
Authors: Chang Huan‐Tsung , Yeung Edward S.
Published: May 2005

Dynamic control in capillary electrophoresis (CE) is able to provide better resolution, shorter separation time, reduced band broadening and better reproducibility for the separations of organic and inorganic ions and large molecules such as proteins and DNA. This article provides an overview of dynamic control in CE by several techniques: pH, temperature, external electric field, and field amplification. These techniques take advantage of the changes in electroosmotic flow coefficient, electrophoretic mobilities of the analytes due to changes in equilibrium, viscosity of the buffer solution, and/or electric field strength. The basic theories and applications to the improvement of the separation performance for standard samples in CE by using these dynamic control techniques are presented. Finally, the advantages and shortcomings of these techniques and the future trends of dynamic control for the separations of real samples are discussed.
Capillary enzymophoresis of nucleic acid fragments using coupled capillary electrophoresis and capillary enzyme microreactors having surface‐immobilized RNA‐modifying enzymes
Authors: Mechref Yehia , El Rassi Ziad
Published: May 2005

This report describes the coupling of capillary enzyme reactors to capillary electrophoresis, which is termed capillary enzymophoresis. In the present study, the capillary enzyme reactors were prepared by immobilizing RNA‐modifying enzymes, e.g., RNase T1 and RNase U2, on the inner walls of 50 μm fused‐silica capillaries. These microreactors served to selectively modify the solutes (or substrates) before entering the separation capillary. Capillary enzymophoresis using single or mixed enzyme reactors proved useful in identifying minute amounts of dinucleotides as well as the fingerprinting of tRNAs. The immobilized RNase T1 and RNase U2 displayed their usual enzymic activities toward RNA fragments and in addition exhibited different activity‐pH dependency than the soluble enzymes. This was attributed to microenvironmental effects arising from the charged nature of the capillary walls in the close proximity of the immobilized enzymes. The enzyme reactors were reusable for several RNA samples and showed chemical and thermal stability

Presented as a part of a lecture (#313) at Pittsburgh Conference 94 (Pittcon '94), Chicago, Illinois, February 27‐March 4, 1994.

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Differential cloning using in‐gel competitive reassociation
Authors: Yokota Hiroshi , Amano Shinya , Yamane Tetsuo , Ataka Kazuto , Kikuya Eriko , Oishi Michio
Published: May 2005

We describe the principle and actual processes of a differential cloning procedure designed for cloning of anonymous restriction DNA fragments whose molecular sizes differ between two genomic DNA preparations from higher organisms as a result of DNA rearrangement, polymorphism, etc. The procedure, which was extensively modified from the original one and still employs in‐gel competitive reassociation (IGCR) as the basic principle, aims for cloning of DNA fragments which exist in one copy or less per mammalian genome. The modified procedure consists of dissociation and reassociation of biotinylated restriction digests of target DNA fragments (from which clones are to be isolated) in the presence of a large excess of reference (competitor) DNA in gel after electrophoresis, which is followed by absorption of the target DNA fragments to streptavidin‐coated tubes and solid‐phase polymerase chain reaction. After repeating these steps we attained substantial enrichment of altered DNA fragments which were originally present in one copy or less per complex mammalian genome.
Rapid chemiluminescent detection of mRNAs on Northern blots with digoxigenin end‐labelled oligonucleotides
Authors: Trayhurn Paul , Duncan Jacqueline S. , Nestor Antoinette , Thomas Moira E. A. , Eastmond Nigel C. , Rayner D. Vernon
Published: May 2005

A simplified, nonradioactive procedure for the detection of specific mRNAs on Northern blots has been developed, utilizing digoxigenin‐labelled oligonucleotides and chemiluminescence. Antisense oligonucleotide (30–35 mer) probes were designed and synthesised based on published cDNA and gene sequences. These probes were end‐labelled (5′) with digoxigenin. Total RNA was fractionated by agarose gel electrophoresis and capillary blotted onto positively charged nylon membranes. After hybridization, the mRNA/digoxigenin‐labelled oligonucleotide complex was detected by a chemiluminescence‐based method using disodium 3‐(4‐methoxyspiro‐[1,2‐dioxetane‐3‐2′(5′chloro)‐tricyclo[3.3.1.13.7]decane]‐4‐yl)phenyl phosphate (CSPD) as substrate. The advantages of this simplified technique for detecting mRNAs in physiological and nutritional studies are described.
Destaining of Coomassie Brilliant Blue R‐250‐stained polyacrylamide gels with sodium chloride solutions
Authors: Sreeramulu Guttapadu , Singh Nagendra K.
Published: May 2005

A novel method for destaining of polyacrylamide gels, stained with Coomassie Brilliant Blue R‐250, is described, based on the use of 0.5 M NaCl in water as the destainer, requiring only 2–3 h. Concentrated (> 2 M) or dilute (< 0.1 M) salt solutions were unsuitable. The method affords the advantage that expensive organic solvents, such as methanol, acetic acid or trichloroacetic acid, are not needed. Furthermore, salt destaining results in darker purple‐blue protein bands as compared to the pale blue color with e.g. methanol/acetic acid destaining.
Studies on the procedure for the construction of cellular protein databases employing micro two‐dimensional electrophoresis: An HL‐60 protein database
Authors: Manabe Takashi , Yamamoto Hideko , Kawai Makoto
Published: May 2005

The procedure to construct a cellular protein database employing a micro two‐dimensional electrophoresis (2‐DE) technique was studied. Protein information on human cultured leukemic cell HL‐60 was collected as an HL‐60 protein database, which consists of a 2‐DE polypeptide map of 670 recognized spots with spot numbering and information on isoelectric point (pI), molecular mass, and percent integrated density. The procedure employed in this report will be applicable for the comparative studies of cellular proteins and will help to construct comprehensive cellular protein databases.
Selective preconcentration for capillary zone electrophoresis using protein G immunoaffinity capillary chromatography
Authors: Cole Laura J , Kennedy Robert T.
Published: May 2005

Capillaries with 150 μm inner diameter were packed with a perfused protein G chromatographic support and used as immunoaffinity preconcentrators for capillary zone electrophoresis. Antibody was loaded onto the protein G support to form an immunoaffinity stationary phase. Injection of samples onto the column caused selective retention and preconcentration of antigen. Injection of appropriate buffers onto the column caused desorption of the antibody and antigen which were then separated by capillary zone electrophoresis. The combination was used on‐line and off‐line. For on‐line combination, a flow‐gated interface coupled the two columns and allowed injection of desorbed zones onto the electrophoresis system. Off‐line coupling required collection of desorbed fractions and then injection onto the electrophoresis system. Flow rates as high as 100 μL/min were used to load sample onto the affinity column. Desorbing flow rates had to be 1 μL/min or less to prevent excessive dilution during desorption. Using the system, 1 mL insulin samples could be loaded onto the affinity column and desorbed in volumes as small as 1 μL for 1000‐fold preconcentration. The use of the preconcentrator with serum samples spiked with insulin was demonstrated.
Charge and size effects in the capillary zone electrophoresis of nuclease A and its variants
Authors: Kálmán Franka , Ma Stacey , Hodel Alec , Fox Robert O. , Horváth Csaba
Published: May 2005

The migration behavior of nuclease A from Staphylococcus aureus and 11 of its variants in capillary zone electrophoresis (CZE) was investigated in the light of their three‐dimensional structure known from X‐ray crystallography and nuclear magnetic resonance (NMR) measurements. Nuclease A (molecular mass 16.8 kDa, pKa 10.3) and the variants differ only in a single amino acid residue and have a very similar crystal structure. With the use of coated quartz capillaries and suitable buffers, the protein migration was investigated at pH from 2.8 to 9.5 without interference by wall adsorption. Although the selectivity of the electrophoretic system for the proteins was mainly determined by their charge differences, certain variants having the same net charge could also be readily separated under nondenaturing conditions. For instance, the mobility of variant K116A was sufficiently higher than that of K116G so that they could be separated by CZE. The structures of both variants are the same except for the solvent‐exposed loop containing residue 116. For this reason, the difference in electrophoretic mobilities can be attributed to the fact that in K116G the backbone of the 112 to 117 amino acids protrudes slightly from the protein, with a concomitant increase in the hydrodynamic radius with respect to that of K116A. Consequently, K116G shows a smaller mobility than K116A due to its larger hydrodynamic radius despite its smaller molecular mass. The interpretation of the experimentally measured mobilities of such closely related proteins therefore requires not only consideration of their electrostatic charge but also the fine details of their molecular structures.
Zinc‐imidazole positive: A new method for DNA detection after electrophoresis on agarose gels not interfering with DNA biological integrity
Authors: Hardy Eugenio , Sosa Angela E. , Pupo Elder , Casalvilla Racmar , Fernandez‐Patron Carlos
Published: May 2005

Nucleic acids separated by gel electrophoresis are commonly detected within the gel matrix with ethidium bromide staining, followed by gel irradiation with ultraviolet (UV) light. When the separated nucleic acids are to be recovered for further characterization or use, this methodology is unsuitable (i) because a significant number of chemical lesions to the nucleic acid molecules are caused, heavily compromising their biological a ctivity, and (ii) because of health hazards due to accumulative direct contact with ethidium bromide and exposure to UV‐light. As an alternative, for preparative purposes, a new non‐toxic detection method employing zinc and imidazole salts is described. After electrophoresis, the gel is first washed in distilled water to substantially remove remaining electrophoresis reagents, then incubated in 40 mM zinc sulfate for 10 min to allow binding of Zn2+ to the DNA, and subsequently washed with distilled water to remove unbound Zn2+ from gel regions devoid of DNA. On soaking in 0.2 M imidazole for a few minutes, zinc‐DNA complexes are visualized as deep‐white (positive) stained bands against a slightly opaque background. The sensitivity is similar to that of ethidium bromide. Gels can be kept in distilled water for months without loss of staining. After zinc chelation, e.g. with EDTA, it is feasible to quantitatively recover chemically intact and biologically active DNA from the gels, as shown by reelectrophoresis and transformation experiments.
Construction of an internal standard for semi‐quantitative polymerase chain reaction analysis of heat shock proteins
Authors: Khan Mahboob A. , Latif Najma , Petrobelli Paola , Yacoub Magdi H. , Dunn Michael J.
Published: May 2005

We describe a simple and effective method for generating competitive cDNA fragments for use as an internal standard in semi‐quantitative polymerase chain reaction (PCR). The use of a nested composite primer to a region slightly within the expected amplicon but with heat shock protein 60 kDa (hsp60) primer sequence appended to the 5′ terminus of the primer produces a “mimic” DNA that will model the PCR kinetics of the target template in sequence context, and PCR primer site, and that will be similar in size. This ensures that both competitor and target template are subjected to similar PCR kinetics and so allows more meaningful quantitation.
pH gradient electrophoresis of basic ribonucleases in sealed slab polyacrylamide gels: Detection and inhibition of enzyme activity in the gel
Authors: Nadano Daita , Yasuda Toshihiro , Sawazaki Kazumi , Takeshita Haruo , Kishi Koichiro
Published: May 2005

A simple method for the separation and specific detection of basic ribonucleases (RNases) was developed. The separation was achieved by polyacrylamide gel electrophoresis in a pH gradient generated by a carrier ampholyte (Pharmalyte 8–10.5) and arginine. In order to prevent interference from atmospheric carbon dioxide, the pH gradient was formed in sealed vertical gel slab. Human nonsecretory‐type RNase, bovine pancreatic RNase A, and other basic proteins could be resolved without expensive equipment or complicated procedures. For activity detection after electrophoresis a zymogram technique was applied, using dry agarose film containing ethidium bromide plus RNA as substrate. This approach affords two advantages: (i) Basic RNase activities can be detected within 15 min, even in crude materials. The sensitivity is better than 0.5 ng of purified human nonsecretory‐type RNase. (ii) An inhibition test of RNase activities in the gel, using human placental‐type RNase inhibitor, can be performed.
Preparative electrophoresis on linear polyacrylamide‐agarose composite gels
Authors: Shainoff John R. , Smejkal Gray B. , Mitkevich Olga , Dibello Patricia M.
Published: May 2005

A preparative method for isolating centigram quantities of high molecular weight polypeptide chains with high resolution and recovery uses linear polyacrylamide/agarose composite (LPAC) gels as electrophoretic media from which the polypeptides can be easily extracted. The composites are prepared in a manner yielding linear copolymers of acrylamide and 1‐allyloxy‐2,3‐propanediol within 2% agarose gels. After electrophoresis in sodium dodecyl sulfate (SDS), protein bands were rapidly visualized for excision by briefly immersing the gel in cold 0.1 M KCl which precipitates the protein‐associated SDS. The gel slices are then freeze‐thawed to disrupt the agarose matrix and promote syneresis of fluid upon centrifugation. The polypeptides are then separated from the polycarylamide in the supernatant solution by precipitating with either acidic isopropanol, trichloroacetic acid, ammonium sulfate or other general protein precipitants. As determined with polypeptide chains of fibrinogen and its cross‐linked derivatives, recoveries were virtually complete (95.4% ± 2.2%), and were independent of molecular weights over the range tested (104–106).
Biochemical changes during regeneration of sunflower (Helianthus annuus L.)
Authors: Martinelli Lucia , Gianazza Elisabetta
Published: May 2005

The main developmental stages in Helianthus annuus organogenesis have been studied in the sunflower hybrid “Giove”. Shoot regeneration was obtained with high efficiency from mature seed cotyledons. Two‐dimensional electrophoresis of protein extracts as well as the isozyme patterns of acid phosphatase, alcohol dehydrogenase, esterase, gluconate‐6‐phosphate dehydrogenase and phosphoglucomutase were compared during growth, callusing and regeneration. Two‐dimensional protein patterns were similar, although polypeptides specific for each developmental phase could be identified. Different 2,4‐dichlorophenoxyacetic acid concentrations or the sampling of specific regions of the seed did not result in significant differences in protein patterns. The activity of alcohol dehydrogenase and phosphoglucomutase appeared very low. For gluconate‐6‐phosphate dehydrogenase no difference, related either to the genotype or to different morphological stages, could be observed; the expression of acid phosphatase varied in a nonsystematic fashion. The isozyme pattern of esterase was related to the genotype as well as to the morphogenic phase.
Preparative affinity membrane electrophoresis
Authors: Horvath Z. Stephen , Gooley Andrew A. , Wrigley Colin W. , Margolis Joel , Williams Keith L.
Published: May 2005

Using the patented Gradiflow system in conjunction with newly developed affinity membranes, the suitability of an electrokinetic technique for affinity fractionation was investigated. Blue dextran incorporated into an affinity membrane was used to deplete a solution of horse serum of albumin, with the result that a majority of serum proteins were enriched tenfold relative to albumin. The technique, when fully developed, would offer some advantages over affinity chromatography, since to a degree it is possible to control which components of the sample are presented to the affinity matrix. Furthermore, the technique would extend the capabilities of the already multifunctional Gradiflow system.
DNA profiling and plant variety registration: 1. The use of random amplified DNA polymorphisms to discriminate between varieties of oilseed rape
Authors: Lee David , Reeves James C. , Cooke Robert J.
Published: May 2005

Before they can be marketed in the UK, newly bred varieties of crop species have to undergo a process of statutory testing, part of which involves the examination of the distinctness, uniformity and stability (DUS) of the variety. DUS testing is also used as the basis for the award of Plant Breeders' Rights. This paper examines the potential of DNA polymorphisms, amplified using arbitrary primers (RAPDs) for use in DUS testing of varieties of oilseed rape. RAPDs using suitable primers can produce high levels of discrimination (> 95%) between varieties, although there are certain problems in gel ‘scoring’ that are only partially resolved by computerised gel scanning/evaluation techniques. Varieties of oilseed rape are also heterogeneous in their RAPD profiles using certain primers, which could cause problems in the DUS testing context. DNA profiling with RAPDs could be used for discrimination between and identification of oilseed rape varieties, but its use for DUS testing needs to be considered carefully.
Fingerprinting of glycans as their 2‐aminoacridone derivatives by capillary electrophoresis and laserinduced fluorescence
Authors: Harland Gary B. , Okafo George , Matejtschuk Paul , Sellick Ian C. , Chapman George E. , Camilleri Patrick
Published: May 2005

Capillary electrophoresis and laser‐induced fluorescence detection have been used to fingerprint the 2‐aminoacridone derivatives of complex glycans released from bovine fetuin and human IgG monoclonal antibodies. The utility of this method in distinguishing between N‐ and O‐linked oligosaccharides and in determining the presence of sialic acid residues in glycan mixtures at an early stage of analysis has been demonstrated.
Electrophoretic separation of recombinant tissue‐type plasminogen activator glycoforms: Validation issues for capillary isoelectric focusing methods
Authors: Moorhouse Kathryn G. , Rickel Carina A. , Chen Anthony B.
Published: May 2005

Attempts were made to validate a capillary isoelectric focusing (cIEF) method for a recombinant glycoprotein as an alternative technique to slab gel isoelectric focusing methods routinely used to monitor such charge heterogeneity. The cIEF method principally separates the charged glycoforms of recombinant tissue‐type plasminogen activator (rt‐PA) on the basis of their sialic acid content. Nine to ten distinct peaks were consistently resolved, with the profile dependent on the class of ampholyte used. The pI of rt‐PA measured with synthetic pI standards was in the range pH 6.5–7.5 with the migration of the standards affected by the presence of the protein. The method showed an acceptable recovery of > 100% and had good sensitivity where 25 ng of protein could be resolved into constituent peaks. Recovery of both major peaks and total protein measured by peak areas was linear over a wide range from 50–1000 μ/mL. A detailed study showed that when a capillary had been used for some time, capillary age affected peak migration times and, to a lesser extent, resolution. Peak migration times were stable over a temperature range of 15–30°C, and decreased predictably with increasing voltages (400–600 V/cm) and decreasing N, N, N′, N′‐tetramethylethylene diamine (TEMED) concentrations (0.4–1.5% v/v). Overall the data indicated that this methodology has the potential to be used in the commercial release of protein pharmaceuticals if variability resulting from capillary age and lot were resolved. Even in its present format the method equals the performance of slab gel IEF whilst offering significant improvements in ease of operation and in time and reagent use.
State of the art of large‐scale genetic purity testing of hybrid vegetable seeds using isoelectric focusing at PetoSluis
Authors: Burg Henk C. J. , van den Berg Bartel M.
Published: May 2005

During the past ten years we have been engaged in developing and applying isoelectric focusing techniques to test the genetic quality of vegetable seeds. We started with isoelectric focusing using carrier ampholytes (IEF‐CA), and continued research with isoelectric focusing in immobilized pH gradients (IEF‐IPG) and two‐dimensional electrophoresis (IPG‐DALT). In addition, we have developed equipment and procedures for large‐scale seed and seedling homogenization, sample preparation and semi‐automatic gel staining. Moreover, we have optimized the sample application and gel running setup for large‐scale analysis. We have developed hybrid purity (inbred) testing methods for all important vegetables, e.g., melon, cole crops, tomato, pepper, watermelon, squash, cucumber, radish etc. using either IEF‐CA or IEF‐IPG of seed or seedling proteins, followed by specific or general protein staining. To indicate the efficiency of the equipment and procedures developed we present results of two of our hybrid purity test methods, namely for brassica using polymorphism for phosphoglucomutase (PGM) from dry seeds, and for tomato using polymorphism for alcohol dehydrogenase (ADH) from imbibed seeds. We show that one person can routinely analyze 1536 individual seeds per day at a cost of about US $0.11 per seed for chemicals, materials, and electrophoresis equipment.
Quantitation of polymerase chain reaction‐amplified DNA fragments by capillary electrophoresis and laserinduced fluorescence detection
Authors: Vincent Ursula , Patra Guy , Therasse Joël , Gareil Pierre
Published: May 2005

Quantitation of DNA fragments amplified by polymerase chain reaction (PCR) is needed for the determination of target DNA in molecular biology. Capillary electrophoresis in entangled polymer solution coupled to laser‐induced fluorescence detection was assessed as an alternative technique to conventional slab gel methods to monitor competitive PCR, which consists of amplifying an internal standard fragment under the same conditions as the target fragment. The fluorescence signal was generated either through end‐labeling of the fragments using 5′‐fluorescein‐labeled primers or through intercalation of ethidium bromide along the double strand. It is shown that the more accurate and reliable results were obtained using this latter pathway.
Subtyping of group‐specific component and protease inhibitor by rapid isoelectric focusing on PhastSystem
Authors: Yaojun Guo , Xinhui Li , Lili Huang
Published: May 2005

A rapid method on PhastSystem was used to investigate the distribution of group‐specific component (GC) and protease inhibitor (PI) subtypes and their gene frequencies from 190 unrelated healthy donors of the Han population in Beijing. Laboratory‐made gels (pH 4.5–5.4 and pH 4.2–4.9) were used for analysis of GC and PI, respectively. Sample loading was 1.5 μL. The separation and visualization time was 0.5 h in each. Gene frequencies were as follows: GC*1F = 0.4891, GC*1S = 0.2432, GC*2 = 0.2678; rare GC variants were discovered in seven cases. The results for PI were; PI*M1=0.7542, PI*M2 = 0.1808, PI*M3 = 0.0650. Good agreement between the observed and expected values in both GC and PI subtyping (for GC, ∑χ2= 1.4043, 0.7<P< 0.8; for PI, ∑χ2= 1.1233, 0.7 <P< 0.8) was obtained.
Sample streaks and smears in immobilized pH gradient gels
Authors: Esteve‐Romero Josep , Simò‐Alfonso Ernesto , Bossi Alessandra , Bresciani Filippo , Righetti Pier Giorgio
Published: May 2005

In immobilized pH gradient (IPG) gel formulations as wide as PH 4–9, encompassing neutrality and containing the pK 7.0 acrylamido buffer as one of the buffering ions, smears are directly proportional to the total amount of the pK 7.0 species. At a total level of 10 mM pK 7.0 in these gel formulations, severe smears occur not only for mildly hydrophobic proteins (e.g., recombinant alcalase and termamylase) but also for the relatively hydrophilic pI marker proteins. Streaks and smears are essentially abolished in recipes devoid of the pK 7.0 compound or in formulations containing a maximum of 3 mM of this component. Although partitioning in water/n‐octanol has shown the pK 7.0 acrylamido buffer to be quite hydrophobic (P = 0.5), the occurrence of smears could be to the presence of oligomers in some commercial preparations. Even when dissolved in n‐propanol, some batches of acrylamido buffers might still contain oligomers, probably formed during the synthetic step.
Novel acrylamido monomers with higher hydrophilicity and improved hydrolytic stability: I. Synthetic route and product characterization
Authors: Simò‐Alfonso Ernesto , Gelfi Cecilia , Sebastiano Roberto , Citterio Attilio , Righetti Pier Giorgio
Published: May 2005

The novel acrylamido monomer reported by our group (N‐acryloylaminoethoxyethanol, AAEE; Chiari et al., Electrophoresis 1994, 15, 177–186), found to combine high hydrophilicity with extraordinary resistance to alkaline hydrolysis, has come under closer scrutiny due to unexpected and random autopolymerization while stored as a 1/1 v/v water solution at 4°C (possibly due to a greater oxidability of the ether group). We have additionally found a unique degradation pathway of the monomer, called “1–6 H‐transfer”, by which the C1 (on the double bond site), by constantly ramming against the C6, next to the ether oxygen (O7, which in fact favors the transfer of the hydrogen atom by C1), produces radicals which more efficiently add to the monomer favoring autopolymerization and cross‐linking. A number of novel monomers is proposed while maintaining the other unique chracteristics of AAEE. One of them, N‐acryloylaminopropanol, offers all the unique, special qualities of AAEE, without the noxious aspects of autopolymerization. Additionally, a synthetic route was optimized, yielding an essentially pure product in a single reaction step, with a yield >99% and an equivalent purity (>99%). The synthesis consists in reacting acryloyl chloride at −40°C in presence of a twofold molar excess of aminopropanol and in ethanol (instead of methanol) as solvent. Other solvents, as well as the use of triethylamine for neutralizing the HCl produced, were found to give a variety of undesired byproducts.
Isolation of proteins and nucleic acids by electrophoresis on disposable gel columns
Authors: Costa Maria A. , Duro Giovanni , Porcasi Rossana , Colombo Paolo , Locorotondo Giovanni , Geraci Domenico , Izzo Vincenzo
Published: May 2005

A simple and cheap one‐step method to isolate proteins or nucleic acids by electrophoresis in disposable gel columns is reported. A disposable syringe was modified to host a gel column and an elution chamber. Starting from a crude extract of E. coli, the laboratory‐made devise allowed the isolation of the maltose binding protein (MBP) fused to a recombinant allergenic molecule with a molecular mass of 58 kDa, from a mixture of several proteins. Also, plasmid DNA could be isolated from a mixture containing chromosomal DNA and RNA, avoiding the use of organic solvents. Electrophoresis was performed at 150 V, 35°C, pH 8.0 and 8.3 for protein and DNA, respectively. The protein or the DNA obtained showed a yield of 80% and a purity grade of 90%, as estimated by densitometry.
Identification of stress proteins in lysates of human cell lines separated by two‐dimensional electrophoresis and electroblotted simulataneously onto two different membranes
Authors: Vogt Christian P. , Willi Annemarie , Hess Daniel , Hunziker Peter E.
Published: May 2005

A protocol based on a combination of established methods for the characterization and identification of inducible stress proteins in human cell lines is described. A particular protein spot, collected from several micropreparative two‐dimensional electrophoresis (2‐DE) gels, is concentrated into a new gel prior to simultaneous electrotransfer onto a Cationic Durapore (CD) membrane and onto a polyvinylidene (PVDF) backup membrane. The protein blotted onto the PVDF support is subjected to N‐terminal sequence analysis. From the protein bound to the CD membrane the peptide mass profile is obtained by proteolytic digestion of the protein followed by the separation of the resulting peptides by high performance liquid chromatography (HPLC) and their detection by on‐line electrospray mass spectrometry (LC/MS). Additional internal sequence information may be obtained by amino acid sequence analysis of peptides collected in the HPLC effluent. The efficiency of this strategy is demonstrated with two proteins extracted from 15 micropreparative 2‐DE gels of an extract of a human liver cell line. The peptide mass fingerprinting of a 60 kDa protein with a pI of 5.3 assigned 22 of 37 peptides to the heat shock protein 60 (Hsp 60). The result was confirmed by the N‐terminal sequence analysis of the undigested protein and of an internal tryptic fragment. The second sample, a 40 kDa protein with a pI of 4.9, was identified as a processed form of the heat shock cognate 71 kDa protein (Hsc 70).