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Keiko Hatae - One of the best experts on this subject based on the ideXlab platform.
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detection of giant myofibrillar proteins connectin and nebulin in fish meat by Electrophoresis in 3 5 gradient sodium dodecyl sulfate polyacrylamide slab gels
Journal of Agricultural and Food Chemistry, 2002Co-Authors: Tomiko Mitsuhashi, Midori Kasai, Keiko HataeAbstract:An improved method was investigated for sodium dodecyl sulfate polyacrylamide slab gel Electrophoresis (SDS−PAGE) to facilitate the analysis of the giant myofibrillar proteins, connectin and nebulin, in fish meat by using jack mackerel (Trachurus japonicus) as the sample fish. It was established that separation of the α-connectin band from the β-connectin band by SDS−PAGE could be achieved by using 3−5% gradient gels with glycerol to facilitate the formation of a gradient with polymerization at 35 °C. SDS−PAGE samples of white dorsal muscle from the jack mackerel were homogenized with a 2% SDS solution containing an inhibitor mixture (1 μg/mL of phenylmethanesulfonyl fluoride, 1 μg/mL of leupeptin, and 1 μg/mL of E-64) and heated at 50 °C for 20 min. Heating these samples at 100 °C for 2 min resulted in the disintegration of connectin but did not affect nebulin. A purified myofibril sample and a whole muscle sample showed similar changes in the overall rate of degradation of whole connectin and nebulin du...
Lloyd M Smith - One of the best experts on this subject based on the ideXlab platform.
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optimization of electric field strength for dna sequencing in capillary gel Electrophoresis
Analytical Chemistry, 1993Co-Authors: John A Luckey, Lloyd M SmithAbstract:: Capillary gel Electrophoresis (CGE) has demonstrated the ability to separate DNA sequencing reactions at speeds up to 25 times as great as conventional slab gel Electrophoresis. These increased speeds are made possible by the efficient heat dissipation of capillaries, which permits higher electric fields to be employed without deleterious thermal effects. The high electric fields, however, also lead to a reduction in the spacing between bands with a concomitant loss of resolution. The resulting tradeoff between speed and resolution is a very important practical aspect of these high-field separations. This work addresses this question by investigating the band broadening and resolution of DNA fragments as they are separated through a fixed distance of gel at field strengths ranging from 50 to 400 V/cm. It is found that the bandwidths of DNA fragments do decrease with the higher field strengths due to a reduction in the diffusional broadening of bands. However, at sufficiently high electric field strengths, the bands begin to broaden again due to the thermal gradient across the gel. This behavior causes the optimum electric field strength for maximum fragment resolution to depend upon the length of fragments being separated. The relative contributions of diffusion and thermal gradients are discussed and used to predict the ultimate performance of constant field capillary gel Electrophoresis.
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optimization of electric field strength for dna sequencing in capillary gel Electrophoresis
Proceedings of SPIE, 1993Co-Authors: John A Luckey, Lloyd M SmithAbstract:Since its development, capillary gel Electrophoresis has demonstrated the ability to separate DNA sequencing reactions at speeds roughly 25 times as great as conventional slab gel Electrophoresis. These increased speeds are the result of using the more efficient dissipation of Joule heating by capillaries. However, to date there have been no studies which quantitate the advantages of disadvantages in operating these gels at high electric field strength. This work addresses this question by investigating the band-broadening of DNA sequencing reactions as they are separated through a fixed distance of gel at field strengths ranging from 50 V/cm to 400 V/cm. It is found that the bandwidths of DNA fragments do decrease with the higher field strengths due to a reduction in diffusional broadening. However, at sufficiently high electric field strengths, the bands begin to broaden again under the influence of an increasing thermal gradient across the diameter of the capillary. The result is an optimum electric field strength in the intermediate range of 100 - 250 V/cm depending on the length of fragments being separated. The relative importance of diffusion and thermal gradients are discussed and used to generate an equation that models the observed band broadening of DNA in capillary gel Electrophoresis (CGE).© (1993) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.
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high speed automated dna sequencing in ultrathin slab gels
Nature Biotechnology, 1992Co-Authors: Anthony J Kostichka, Michael Marchbanks, Robert L Brumley, Howard Drossman, Lloyd M SmithAbstract:Publisher Summary The demands of the human genome initiative for improved DNA sequencing technology are considerable. At this time, virtually all DNA sequencing is based on the separation of DNA fragments in high-resolution polyacrylamide gels. One reasonable approach to improved sequencing is, thus, to increase the performance of such gel-based sequencing methods. To increase the overall throughput of automated sequencing instruments, by a comparable factor, it is necessary to obtain a similar parallelism, in conjunction with these extremely rapid separations. There are two obvious approaches to this problem: (1) employ many gel-filled capillaries in parallel or (2) perform the separations in a slab gel format. This chapter describes the instrumentation and methods that have been developed for high-speed automated DNA sequencing, by ultrathin slab gel Electrophoresis. A sketch of the system is discussed in detail in this chapter. It consists of a horizontal gel Electrophoresis cell, a laser, and optical components to provide a line of fluorescence excitation across the gel near its bottom, collection optics, a charge-coupled device (CCD) detector system to measure the emitted fluorescence, and a computer system to control the CCD camera as well as to store and analyze the resultant data.
Huantsung Chang - One of the best experts on this subject based on the ideXlab platform.
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capillary electropherograms for restriction fragment length polymorphism of helicobacter pylori
Electrophoresis, 2008Co-Authors: Mingjong Bair, Chiulin Chen, Chengkang Chiang, Mingfeng Huang, Huantsung ChangAbstract:Rapid identification of Helicobacter pylori strains is of importance for diagnosis and then treatment of duodenal and gastric ulcers. We developed a CE approach for the analysis of RFLP of the PCR products of urease (UreAB) gene and flagellin A (FlaA) gene fragments. Prior to CE analysis, the 2.4-kbp UreAB and 1.5-kbp FlaA PCR products were digested with the restriction enzymes HaeIII and HhaI, respectively. The DNA fragments were then separated by CE in conjunction with laser-induced fluorescence detection using poly(ethylene oxide) in the presence of electroosmotic flow. The DNA fragments range in sizes 259-1831 bp and 12-827 bp for UreAB and FlaA restriction fragments, respectively. Of 27 samples, the CE approach provided five and ten different RFLP patterns of the HaeIII and HhaI digests. The RFLP of PCR products of the two genes allow great sensitivity of identification of H. pylori strains. When compared with slab gel Electrophoresis, the present CE approach provides advantages of rapidity (within 6 min per run), simplicity, and automation. The preliminary results have shown great practicality of the CE approach for screening H. pylori strains.
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capillary electrophoretic restriction fragment length polymorphism patterns for the mycobacterial hsp65 gene
Journal of Clinical Microbiology, 2004Co-Authors: Poling Chang, Chiachien Hung, Huantsung ChangAbstract:PCR-restriction fragment length polymorphism (RFLP) analysis is a nonprobe method for the rapid identification of Mycobacterium species. We demonstrate the separation of DNA or restriction fragments digested from the mycobacterial gene encoding the 65-kDa heat shock protein (hsp65) by capillary Electrophoresis (CE). By using a pair of unlabeled primers, Tb11 and Tb12, and only one restriction enzyme, HaeIII, we investigated a total of 52 reference and clinical strains encompassing 12 Mycobacterium species. The electrophoretic separation of high-resolution CE required <20 min and was capable of identifying fragments as small as 12 bp. A good agreement of measurement was observed between the sizes of restriction fragments resolved by CE, and the real sizes were deduced from the sequence analysis. Distinct differentiations were also well demonstrated between some species and subspecies by an extra HaeIII digestion site. With the advantage of the complete RFLP pattern available from CE, it appears to be more convenient to use an electropherogram rather than performing the cumbersome slab gel Electrophoresis plus diagnostic algorithm to identify Mycobacterium species. Beyond the agarose and polyacrylamide gel Electrophoresis, high-resolution CE provides an alternative for rapid identification of Mycobacterium species that is feasible for automation and routine use without the need for costly probes.
Tomiko Mitsuhashi - One of the best experts on this subject based on the ideXlab platform.
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detection of giant myofibrillar proteins connectin and nebulin in fish meat by Electrophoresis in 3 5 gradient sodium dodecyl sulfate polyacrylamide slab gels
Journal of Agricultural and Food Chemistry, 2002Co-Authors: Tomiko Mitsuhashi, Midori Kasai, Keiko HataeAbstract:An improved method was investigated for sodium dodecyl sulfate polyacrylamide slab gel Electrophoresis (SDS−PAGE) to facilitate the analysis of the giant myofibrillar proteins, connectin and nebulin, in fish meat by using jack mackerel (Trachurus japonicus) as the sample fish. It was established that separation of the α-connectin band from the β-connectin band by SDS−PAGE could be achieved by using 3−5% gradient gels with glycerol to facilitate the formation of a gradient with polymerization at 35 °C. SDS−PAGE samples of white dorsal muscle from the jack mackerel were homogenized with a 2% SDS solution containing an inhibitor mixture (1 μg/mL of phenylmethanesulfonyl fluoride, 1 μg/mL of leupeptin, and 1 μg/mL of E-64) and heated at 50 °C for 20 min. Heating these samples at 100 °C for 2 min resulted in the disintegration of connectin but did not affect nebulin. A purified myofibril sample and a whole muscle sample showed similar changes in the overall rate of degradation of whole connectin and nebulin du...
Seong Ho Kang - One of the best experts on this subject based on the ideXlab platform.
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voltage programming based capillary gel Electrophoresis for the fast detection of angiotensin converting enzyme insertion deletion polymorphism with high sensitivity
IEEE Journal of Solid-state Circuits, 2016Co-Authors: Seong Ho KangAbstract:A voltage-programming-based capillary gel Electrophoresis method with a laser-induced fluorescence detector was developed for the fast and highly sensitive detection of DNA molecules related to angiotensin-converting enzyme insertion/deletion polymorphism, which has been reported to influence predisposition to various diseases such as cardiovascular disease, high blood pressure, myocardial infarction, and Alzheimer's disease. Various voltage programs were investigated for fast detection of specific DNA molecules of angiotensin-converting enzyme insertion/deletion polymorphism as a function of migration time and separation efficiency to establish the effect of voltage strength to resolution. Finally, the amplified products of the angiotensin-converting enzyme insertion/deletion polymorphism (190 and 490 bp DNA) were analyzed in 3.2 min without losing resolution under optimum voltage programming conditions, which were at least 75 times faster than conventional slab gel Electrophoresis. In addition, the capillary gel Electrophoresis method also successfully applied to the analysis of real human blood samples, although no polymorphism genes were detected by slab gel Electrophoresis. Consequently, the developed voltage-programming capillary gel Electrophoresis method with laser-induced fluorescence detection is an effective, rapid analysis technique for highly sensitive detection of disease-related specific DNA molecules.
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fast parallel detection of feline panleukopenia virus dna by multi channel microchip Electrophoresis with programmed step electric field strength
IEEE Journal of Solid-state Circuits, 2013Co-Authors: Seong Ho KangAbstract:A multi-channel microchip Electrophoresis using a programmed step electric field strength (PSEFS) method was investigated for fast parallel detection of feline panleukopenia virus (FPV) DNA. An expanded laser beam, a 10× objective lens, and a charge-coupled device camera were used to simultaneously detect the separations in three parallel channels using laser-induced fluorescence detection. The parallel separations of a 100-bp DNA ladder were demonstrated on the system using a sieving gel matrix of 0.5% poly(ethylene oxide) (M(r) = 8 000 000) in the individual channels. In addition, the PSEFS method was also applied for faster DNA separation without loss of resolving power. A DNA size marker, FPV DNA sample, and a negative control were simultaneously analyzed with single-run and one-step detection. The FPV DNA was clearly distinguished within 30 s, which was more than 100 times faster than with conventional slab gel Electrophoresis. The proposed multi-channel microchip Electrophoresis with PSEFS was demonstrated to be a simple and powerful diagnostic method to analyze multiple disease-related DNA fragments in parallel with high speed, throughput, and accuracy.
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Ultra-fast Detection and Differentiation of Mycoplasma haemofelis and Candidatus M. Haemominutum in Korean Feral Cats by Microchip Electrophoresis with Programmed Field Strength Gradients
Bulletin of The Korean Chemical Society, 2008Co-Authors: Kailasa Suresh Kumar, Hee Gu Lee, Dong Jin Yoo, Seong Ho KangAbstract:A microchip-based capillary gel Electrophoresis (MCGE) technique was developed for the ultra-fast detection and differentiation of Candidatus Mycoplasma haemominutum (Candidatus M. haemominutum, California strain) and Mycoplasma haemofelis (M. haemofelis, Ohio strain) in Korean feral cats through the application of programmed field strength gradients (PFSG) in a conventional glass double-T microchip. The effects of the poly (ethyleneoxide) (PEO) concentration and electric field strength on the separation of DNA fragments were investigated. The PCR-amplified products of Candidatus M. haemominutum (202-bp) and M. haemofelis (273bp) were analyzed by MCGE within 75 s under a constant applied electric field of 117.6 V/cm and a sieving matrix of 0.3% PEO (Mr 8 000 000). When the PFSG was applied, MCGE analysis generated results 6.8-times faster without any loss of resolution or reproducibility. The MCGE-PFSG technique was also applied to eleven samples selected randomly from 33 positive samples. The samples were detected and differentiated within 11 s. The analysis time of the MCGE-PFSG technique was approximately 980-times faster than that using conventional slab gel Electrophoresis.