Applied and theoretical electrophoresis : the official journal of the International Electrophoresis Society最新文献

{"title":"Benefits of advanced gel electrophoresis data analysis methods.","authors":"D Tietz","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Significantly more information can be obtained if sample mobilities are measured at several gel concentrations (%T) and evaluated on the basis of a mathematical-physical model. This allows one to exploit differences in particle size, charge and shape, and results in the following advantages: (i) Charge and size isomers of proteins can be detected. It is possible to separate components of similar size, but different surface net charge density. (ii) Samples yielding patterns with no discernible peaks can be evaluated to produce size and charge distribution profiles, as shown for protein-conjugated meningitis vaccines. Particle distributions are calculated from 2-D Serwer-type gels. (iii) Different DNA conformations are recognized and reasonable size estimates are available for anomalously migrating DNA species (e.g., bent kinetoplast DNA) or DNA complexes (nucleosome core particles).</p>","PeriodicalId":77007,"journal":{"name":"Applied and theoretical electrophoresis : the official journal of the International Electrophoresis Society","volume":"5 2","pages":"107-11"},"PeriodicalIF":0.0,"publicationDate":"1995-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19554075","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A chemiluminescence-based detection system for human DNA quantitation and restriction fragment length polymorphism (RFLP) analysis.","authors":"A M Giusti,&nbsp;B Budowle","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>A protocol for the chemiluminescent detection of restriction fragment length polymorphism (RFLP) profiles with the sensitivity of radioisotope-based detection systems is presented. RFLP profiles for the loci D2S44, D17S79, D1S7, D4S139, and D5S110 were obtained from ten nanograms of HaeIII-digested K562 DNA and 50 nanograms of human genomic DNA isolated from bloodstains. DNA is transferred to an amphoteric membrane using a neutral, high salt transfer procedure. Oligonucleotide probes directly conjugated to alkaline phosphatase are hybridized to the membranes, then the membranes are incubated with a substrate that emits light upon chemical cleavage by the alkaline phosphatase. Several variables were investigated to determine the optimum conditions for RFLP profile generation, among them transfer conditions, film selection, and exposure conditions. A chemiluminscent human DNA quantitation protocol, consistent with the RFLP detection protocol, is also presented.</p>","PeriodicalId":77007,"journal":{"name":"Applied and theoretical electrophoresis : the official journal of the International Electrophoresis Society","volume":"5 2","pages":"89-98"},"PeriodicalIF":0.0,"publicationDate":"1995-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19553997","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of crosslinked polyamines suitable for synthesizing complex ampholytes for isoelectric focusing.","authors":"D R McWhinney,&nbsp;P DeShong,&nbsp;L S Rodkey","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Commercially available ampholytes used in isoelectric focusing applications vary widely from source to source in their resolving power. This study was initiated to develop alternative ampholyte formulations for high resolution preparative and analytical isoelectric focusing. Initial IR spectroscopy studies showed that divalent acid esters would efficiently crosslink available polyamines with complete consumption of ester. Fast atom bombardment mass spectroscopic analysis of resulting crosslinked polyamines showed extensive structural heterogeneity of the resulting polyamine mixture. Conversion of the polyamine mixture to functional zwitterions using alpha,beta-unsaturated carboxylic acids yielded mixtures giving smooth pH gradients in acrylamide gel isoelectric focusing. Further analysis of these mixtures in immobilized pH gradients showed increases in heterogeneity of available carrier species over similar zwitterion mixtures made using only commercially available polyamine monomers. The mixtures were also more heterogeneous than commercially available ampholytes when analyzed by picric acid precipitation in immobilized pH gradients.</p>","PeriodicalId":77007,"journal":{"name":"Applied and theoretical electrophoresis : the official journal of the International Electrophoresis Society","volume":"4 4","pages":"167-73"},"PeriodicalIF":0.0,"publicationDate":"1995-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18604436","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Time course and inhibitors of Hae III digestion in the forensic laboratory.","authors":"E A Benzinger,&nbsp;R E Shirley,&nbsp;A K Riech,&nbsp;P J Sallee,&nbsp;K L Boster,&nbsp;K C Lumney","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Forensic restriction fragment length polymorphism (RFLP) profiling has become standardized in the United States and Canada to the extent that all federal, state and local forensic laboratories employ the restriction enzyme Hae III. Forensic DNA samples are sometimes resistant to digestion with Hae III. This problem is typically ascribed to sample characteristics and is dealt with through prolonged incubation with Hae III, repeated incubations and/or re-extraction. We determined and extensively tested the maximum useful duration of Hae III incubation for several types of samples. We also examined the effect of common DNA extraction reagents and laboratory procedures on subsequent restrictability of DNA. No benefit whatsoever was found to continuing digestion reactions past 1 hour. Certain laboratory reagents were found to inhibit Hae III activity at extremely low concentrations whereas others were found to have little effect on the success of the restriction digestion. Significantly, no contaminating reagent or other laboratory procedure was found to cause loss of Hae III specificity, whether enzyme activity was compromised or not. The results of this study allow the streamlining of the RFLP procedure and provide a basis for understanding how laboratory procedures may adversely affect Hae III activity and DNA digestion.</p>","PeriodicalId":77007,"journal":{"name":"Applied and theoretical electrophoresis : the official journal of the International Electrophoresis Society","volume":"4 4","pages":"179-88"},"PeriodicalIF":0.0,"publicationDate":"1995-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18604437","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Separation, real-time migration monitoring and selective zone retrieval using a computer controlled system for automated analysis.","authors":"E Gombocz,&nbsp;E Cortez","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>High throughput routine analysis of dsDNA fragments or molecular weight determination of proteins via electrophoresis still require significant efforts to obtain results of high reproducibility and accuracy. This paper analyses the use of a fully automated multi-purpose real-time gel electrophoresis system in these applications and evaluates the benefits of this new concept for routine and research. By comparing currently used systems with this new approach, it also addresses the analytical use of information resulting from real-time dynamic migration monitoring via fluorescence photometry over commonly obtained results from post-run fixation, visualization and evaluation at a single time of the separation. The simultaneous separation of components in multi-gel systems, pre-concentration of sample components, and the ability to perform in-gel assays for biological activity are discussed on basis of routine gel separations of restriction enzyme digested DNA fragments, native and denaturing protein separations and enzyme activity determination. Interactive, selective retrieval of separated components in the nano- to microgram range is carried out for real-time isolation of proteins or dsDNA fragments. Results are compared to blotting in respect to ease of use and transfer efficiency and for immediate availability of macromolecules for sequencing or mass spectroscopy. The Windows-based operating software is critically reviewed for functionality, user-friendliness, graphical data representation and GLP compliance for LIMS oriented forensic or certified laboratories. A statistical evaluation of lane-to-lane and gel-to-gel reproducibility of mobility data, quantification and molecular weight determination concludes the paper.</p>","PeriodicalId":77007,"journal":{"name":"Applied and theoretical electrophoresis : the official journal of the International Electrophoresis Society","volume":"4 4","pages":"197-209"},"PeriodicalIF":0.0,"publicationDate":"1995-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18604926","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluation of the specificity of alkaline phosphatase-conjugated oligonucleotide probes for forensic DNA analysis.","authors":"E A Benzinger,&nbsp;A K Riech,&nbsp;R E Shirley,&nbsp;K R Kucharik","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Current methods of forensic DNA profiling by restriction fragment length polymorphism analysis rely on radioactive detection of DNA. The use of radioactive isotopes is complicated, expensive and requires elaborate safety precautions. Recently, non-radioactive detection methods involving the use of alkaline phosphatase-conjugated oligonucleotide probes have become available. These probes differ from most 32P-labeled probes in that they are synthetic oligonucleotides, whereas the 32P-labeled probes are purified plasmid inserts from cloned VNTR regions. Because of this difference, it is possible that the specificity of the non-isotopic probe will differ from that of the 32P-labeled probe. This study compares the specificity of the two types of probes by parallel analysis of a set of DNA samples, including a subset of relatively small alleles, at the loci D1S7, D2S44, D4S139, D10S28, and D17S26. We found that although the alkaline phosphatase detection method is slightly less sensitive than 32P detection, the AP-conjugated oligonucleotide probes tested have specificity comparable to, and are appropriate and suitable substitutes for, 32P-labeled plasmid inserts.</p>","PeriodicalId":77007,"journal":{"name":"Applied and theoretical electrophoresis : the official journal of the International Electrophoresis Society","volume":"4 4","pages":"161-5"},"PeriodicalIF":0.0,"publicationDate":"1995-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18604435","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The analysis of simple sequence repeat DNA in soybean by Capillary Gel Electrophoresis.","authors":"M A Marino, L A Turni, S A Del Rio, P E Williams, P B Cregan","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>The objective of this work is to examine the presence of simple sequence repeat (SSR) DNA in soybean plant genotypes by Capillary Gel Electrophoresis (CGE). The SSR DNA length polymorphism in soybean determines the variation in polymerase chain reaction (PCR) product lengths. Loci were chosen where amplification produced one PCR product per genotype (M.S. Akkaya et al., 1992). The F1 hybrids of parents carrying different alleles produced two PCR products identical to the two parents. The CGE system used a 3%T,3%C polyacrylamide gel capillary with an effective length of 40 cm. The PCR products with lengths of 150 to 200 base pairs were monitored at 260 nm. The analysis time was under 50 minutes. CGE is capable of separating these PCR products by base pair number the same as conventional sequencing gel techniques. CGE offers an automated, high speed, high resolution analytical method for determining soybean SSR allele sizes as compared with the traditional methodologies.</p>","PeriodicalId":77007,"journal":{"name":"Applied and theoretical electrophoresis : the official journal of the International Electrophoresis Society","volume":"5 1","pages":"1-5"},"PeriodicalIF":0.0,"publicationDate":"1995-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19516233","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Altered protein synthesis in p53 null and hemizygous transgenic mouse embryonic fibroblasts.","authors":"C He,&nbsp;B A Merrick,&nbsp;R M Patterson,&nbsp;J K Selkirk","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Embryonic fibroblasts derived from p53-deficient transgenic mice showed distinct phenotypic and biological changes in vitro. In this study, we investigated the possible impact of p53 on the synthesis of other cellular proteins by comparing the protein profiles of p53 null (-/-), hemizygous (+/-) and p53 positive homozygous (+/+) cells using high resolution two dimensional gel electrophoresis. A total of more than 850 proteins were detected in each cell line labeled with 35S-methionine by using computerized image analysis, and a number of proteins were detected with qualitative or quantitative changes in p53-/- cells and to a lesser extent in p53+/- cells. Specifically, seven proteins became undetectable, and no new proteins were detected in p53-/- cells. Neither newly expressed nor absent proteins were detected in p53+/- cell line. Quantitatively, a total of 97 and 59 proteins were detected with significant quantitative changes (3 fold or greater) in p53-/- and p53+/- cells, respectively. Generally, most protein changes fell into one of the following four patterns: 1) progressively decreased synthesis in cells from p53+/+ to p53+/- to p53-/- cells; 2) progressively increased synthesis in cells from p53+/+ to p53+/- to p53-/- cells; 3) decreased synthesis only in p53-/- cells; and 4) increased synthesis only in p53-/- cells. A 70 kD heat shock protein (Hsp 70) was identified and showed a greater than 1,000-fold increase in p53-/- cells compared to that in p53+/+ cells. Transferrin, tropomyosin, and proliferating cell nuclear antigen (PCNA) have also been identified and measured in this study. Synthesis of transferrin and tropomyosin was significantly increased or decreased, respectively in p53-/- cells, whereas expression of PCNA showed no significant change in p53-/- cells despite their much higher (3-4 times) proliferation rate than the other two cell lines (p53+/+ and p53+/- cells). We conclude that disruption of a single important gene, p53, results in a cascade of protein changes which are related to the loss of p53 mediated negative growth effects on cell cycle control.</p>","PeriodicalId":77007,"journal":{"name":"Applied and theoretical electrophoresis : the official journal of the International Electrophoresis Society","volume":"5 1","pages":"15-24"},"PeriodicalIF":0.0,"publicationDate":"1995-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19516234","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Molecular sieving by polymer solutions: dependence on particle and polymer size, independence of polymer entanglement.","authors":"S P Radko,&nbsp;A Chrambach","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>The previous postulate of collision and displacement mechanisms of molecular sieving based on a biphasic plot of retardation coefficient vs particle radius (\"R-plot\") was extended and modified in four ways: i) A wider size range of particles and polymers confirmed the biphasic nature of the R-plot and, in addition, revealed a third mechanistic phase in the largest size range of particles and polymers which exhibits a positive slope in plots of retardation coefficient vs log (particle or polymer size), presumably denoting a collision mechanism. ii) Peaks of retardation in polyethyleneglycol (PEG) solutions were found with a particle M(r) of 10(7) independently of the M(r) of PEG, and with a PEG M(r) of 4 x 10(5) independently of particle M(r), showing that the retardation mechanism is not qualitatively a function of the particle/polymer size ratio as postulated previously, although quantitatively retardation is directly related to the size of particle and polymer. iii) Items i) and ii) were confirmed using band width in lieu of mobility measurement. iv) The entanglement threshold, c*, was found to decrease monotonically across the entire polymer size range in which the triphasic retardation takes place. Thus c* cannot be the sole cause for a non-monotonic change of retardation or normalized relative bandwidth with polymer size and particle size. Moreover, Ferguson plots across c* do not reflect it in any way.</p>","PeriodicalId":77007,"journal":{"name":"Applied and theoretical electrophoresis : the official journal of the International Electrophoresis Society","volume":"5 2","pages":"79-87"},"PeriodicalIF":0.0,"publicationDate":"1995-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19553996","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A desktop, low-cost video fluorometer for quantitation of macromolecules after gel electrophoresis.","authors":"G A Griess,&nbsp;D Louie,&nbsp;P Serwer","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>After gel electrophoresis, quantification of in-gel fluorescence is sometimes used to assay the macromolecules fractionated. Both procedures and equipment that have the following improvements are presented here for direct video fluorometry of gels used for electrophoresis: comparatively low cost, high ease of use and low consumption of space. This equipment has a linear response to the amount of ethidium-stained DNA that forms a band in an agarose gel.</p>","PeriodicalId":77007,"journal":{"name":"Applied and theoretical electrophoresis : the official journal of the International Electrophoresis Society","volume":"4 4","pages":"175-7"},"PeriodicalIF":0.0,"publicationDate":"1995-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"18547026","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}