共查询到14条相似文献,搜索用时 62 毫秒
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目的:建立从转基因作物中快速提取DNA的方法.方法 :采用Chelex-100法提取抗草甘膦大豆和非转基因大豆、转基因抗虫玉米Bt176和非转基因玉米中的DNA,使用PCR扩增大豆和玉米的内源基因(Lectin,zSSⅡb)及外源特异性序列(CaMV35S,Bt176)评价提取核酸的质量.结果 :Chelex-100法能够快速在1h之内从大豆和玉米中提取DNA,所提取的DNA可以直接用于PCR扩增反应,PCR扩增产物电泳条带清晰,转基因抗草甘膦大豆样品和转基因抗虫玉米Bt176检测均出现强阳性结果.结论 :Chelex-100法提取DNA可以作为转基因检测的模板,该方法具有经济、简便、快速的特点,适合于转基因检测工作. 相似文献
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《Nucleosides, nucleotides & nucleic acids》2013,32(6-7):1057-1064
Contiguous stacking hybridization of oligodeoxyribonucleotides with a stem of preformed minihairpin structure of a DNA template was studied with the use of UV‐melting technique. It was shown that the free‐energy of the coaxial stacking interaction (ΔG°ST at 37°C, 1 M NaCl, pH 7.4) at the complementary interface XA*pTY/ZATV (an asterisk stands for a nick) strongly depends on the type of nearest neighbor bases X and Y flanking the nicked dinucleotide step. The maximum efficiency of the coaxial stacking was observed for the PuA*pTPy/PuATPy interface, whereas the minimum efficiency was obtained for the PyA*pTPu/PyATPu interface. A 5′‐phosphate residue in the nick enhances the coaxial stacking. In dependence on duplex structure the observed efficiency of A*T/AT coaxial stacking varied from (? 0.97 kcal/mol) for unphosphorylated TA*TA/TATA interface to three‐fold higher value (? 2.78 kcal/mol) for GA*pTT/AATC interface. 相似文献
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Mitchelson KR 《Molecular biotechnology》2003,24(1):41-68
Capillary electrophoresis has advanced enormously over the last 10 yr as a tool for DNA sequencing, driven by the human and
other major genome projects and by the need for rapid electrophoresis-based DNA diagnostic tests. The common need of these
analyses is a platform providing very high throughput, high-quality data, and low process costs. These demands have led to
capillary electrophoresis machines with multiple capillaries providing highly parallel analyses, to new electrophoresis matrices,
to highly sensitive spectrofluorometers, and to brighter, spectrally distinct fluorescent dyes with which to label DNA. Capillary
devices have also been engineered onto microchip formats, on which both the amount of sample required for analysis and the
speed of analysis are increased by an order of magnitude. This review examines the advances made in capillary and chip-based
microdevices and in the different DNA-based assays developed for mutation detection and genotype analysis using capillary
electrophoresis. The automation of attendant processes such as for DNA sample preparation, PCR, and analyte purification are
also reviewed. Together, these technological developments provide the throughput demanded by the large genome-sequencing projects. 相似文献
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Repetitive sequences in DNA molecules, some of which are palindromic, tend to form stable cruciforms. These are frequently located in promoter regions of a specific operon and origin of replication. Temperature gradient gel electrophoresis can be used to distinguish among various supercoiled DNA topoisomers and to ascertain whether or not the cruciform motif has been extruded. In the current study, this technique is implemented for the first time to address the role of temperature in cruciform extrusion from plasmids. 相似文献
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DNA amplification technology has been applied to clinical diagnosis of infectious disease, genetic disorder, and cancer. After
in vitro amplification of a particular DNA region, the methods of analysis for these amplified samples play a pivotal role
in clinical diagnosis. Conventional gel electrophoresis has been routinely used in the lab for checking DNA. The whole procedure
is time consuming and requires more than 1 ng of DNA for detection. To achieve greater performance in DNA diagnosis, we demonstrated
capillary electrophoresis with laser induced fluorescence detection for analysis of amplified DNA. The analysis of DNA could
be completed within 3 min and the data is directly entered into the computer. Considering the automatic and rapid process,
we believe that this method could be routinely utilized for the clinical diagnosis of amplified DNA products. 相似文献
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Detection of single-copy genes in DNA from transgenic plants by nonradioactive Southern blot analysis 总被引:1,自引:0,他引:1
Matthew S. McCabe J. Brian Power Ad M. M. de Laat Michael R. Davey 《Molecular biotechnology》1997,7(1):79-84
Improvements to the sensitivity, speed, and reproducibility of digoxigenin (DIG)-labeled probes and chemiluminescent substrates
makes these compounds increasingly popular to detect nucleic acids. High sensitivity and low background are essential in Southern
blot analysis, particularly with plant DNA. This article describes a nonradioactive system to detect single-copy genes in
transgenic plants. Labeling using the polymerase chain reaction (PCR) was employed to produce highly sensitive and reusable
DIG-labeled probes. The background was reduced by immobilizing the DNA onto nylon filters by alkaline transfer and by minimized
gel handling; the signal-to-noise ratio was improved by modification of the detection procedure. 相似文献
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Suqin Han 《Luminescence》2005,20(6):405-410
A simple and rapid capillary electrophoresis with direct chemiluminescence method has been developed for the determination of five natural pharmacologically active compounds including rutin, protocatechuic aldehyde, chlorogenic acid, luteolin and protocatechuic acid. The luminol as a component of the separation electrolyte buffer was introduced at the head of the separation capillary. The separation of five compounds was carried out in a fused-silica capillary with 15.0 mmol/L tetraborate, 1.0 mmol/L SDS and 0.42 mmol/L luminol (pH 8.5). The analytes was determined by enhancing the chemiluminescence of luminol with 0.13 mmol/L K3Fe(CN)6 in 0.05 mol/L NaOH, which was introduced at the post-column stage. The voltage applied was 16 kV. Under the optimum conditions, the analytes were separated within 10 min. The excellent linearity was obtained over two to three orders of magnitude with a detection limit (signal:noise = 3) of 0.012-0.055 micromol/L for all five analytes. The method was successfully used in the analysis of pharmaceutical and biological samples, and the assay results were satisfactory. 相似文献
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Krylova SM Karkhanina AA Musheev MU Bagg EA Schofield CJ Krylov SN 《Analytical biochemistry》2011,(2):8617-265
The AlkB family of oxygenases catalyze the removal of alkyl groups from nucleic acid substrates in an iron and 2-oxoglutarate-dependent manner and have roles including in DNA repair. To understand the biological functions of these DNA-dealkylating enzymes it is desirable to measure their expression levels in vitro and in vivo in complex biological matrixes. Quantitative analyses of the enzymes require affinity probes capable of binding AlkB family members selectively and with high affinity. Here we report that DNA aptamers can serve as efficient affinity probes for quantitative detection of such enzymes in vitro. Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) was applied as a general tool for: (i) selection of DNA aptamers, (ii) characterization of binding parameters for the aptamers, and (iii) quantitative detection of the target in an aptamer-based affinity analysis. The selected aptamers have a range of Kd values between 20 and 240 nM. The aptamers enabled accurate quantitative analysis of AlkB even in the presence of the Escherichia coli cell lysate. Aptamers can likely be developed for other nucleic acid repair enzymes. They may also be developed for use in in vitro and potentially in vivo studies of known nucleic acid-modifying enzymes including for functional analysis. 相似文献