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41.
中国人CD59cDNA克隆、序列分析及其表达 总被引:1,自引:0,他引:1
用RT-PCR方法从中国人胚胎总mRNA中扩增出420bp人补体终末阶段同源限制因子(CD59)的cDNA,序列分析结果表明,所获得的CD59cDNA与文献报道中的基因序列完全一致,含CD59全长编码区.构建了真核表达质粒,在3T3细胞中得到了表达. 相似文献
42.
提出一种无胶毛细管电泳分离碱基对范围宽的DNA片段的方法.用DB-1气相色谱毛细管柱,以羟乙基纤维素为筛分介质,研究了λDNA/EcoRⅠ+HindⅢ限制性片段的分离,分离效率达1.2×106板/m,检测限为2.1×10-17mol.应用于一种鲤鱼种族鉴别基因的聚合酶链反应(PCR)扩增产物的分离鉴定,结果与实际相符,分离速度比传统电泳方法提高20倍. 相似文献
43.
44.
We present a mathematical algorithm for the analysis of electrophoretic patterns resulting from arbitrarily primed PCR profiling. The algorithm is based on the established mathematical procedures applied to the analysis of digital images of gel patterns. The algorithm includes (a) transformation of the image into a matrix form, (b) identification of every electrophoretic lane as a set of matrix columns that are further mathematically processed, (c) averaging of matrix columns corresponding to electrophoretic lanes that define lane representatives, (d) elimination of "smiling" bands, (e) solving the problem of a lane offset, and (f) removal of the background. Representation of individual electrophoretic lanes in the form of functions allows interlane comparisons and further mathematical analysis. Direct comparison of selected lanes was obtained by employing correlation analysis. Gel images were those obtained after arbitrarily primed PCR analysis of DNA that underwent damage induced by gamma radiation from a (60)Co source. The applied method proved to be useful for elimination of subjectivity of visual inspection. It offers the possibility to avoid overlooking important differences in case of suboptimal electrophoretic resolution. In addition, higher precision is achieved in the assessment of quantitative differences due to better insight into experimental artifacts. These simple mathematical methods offer an open-type algorithm, i.e., this algorithm enables easy implementation of different parameters that may be useful for other analytical needs. 相似文献
45.
Nakayama T Kurosawa Y Furui S Kerman K Kobayashi M Rao SR Yonezawa Y Nakano K Hino A Yamamura S Takamura Y Tamiya E 《Analytical and bioanalytical chemistry》2006,386(5):1327-1333
Polymerase chain reaction (PCR) is an essential part of research based on genomics or cell analysis. The development of a
microfluidic device that would be suitable for high-temperature-based reactions therefore becomes an important contribution
towards the integration of micro-total analysis systems (μTAS). However, problems associated with the generation of air bubbles
in the microchannels before the introduction of the assay liquid, which we call the “initial start-up” in this study, made
the flow irregular and unstable. In this report, we have tried to address these problems by adapting a novel liquid-flow method
for high-temperature-based reactions. A PDMS-based microfluidic device was fabricated by soft-lithography techniques and placed
on a cartridge heater. The generation of the air bubbles was prevented by introducing the fluorinated oil, an inert and highly
viscous liquid, as the cap just before the introduction of the sample solutions into the microchannels. The technique was
applied for continuous-flow PCR, which could perform PCR on-chip in a microfluidic system. For the evaluation of practical
accuracy, plasmid DNA that serves as a reference molecule for the quantification of genetically modified (GM) maize was used
as the template DNA for continuous-flow PCR. After PCR, the products were collected in a vial and analyzed by gel electrophoresis
to confirm the accuracy of the results. Additionally, quantitative continuous-flow PCR was performed using TaqMan technology
on our PCR device. A laser detection system was also used for the quantitative PCR method. We observed a linear relationship
between the threshold cycle (Ct) and the initial DNA concentration. These results showed that it would be possible to quantify
the initial copies of the template DNA on our microfluidic device. Accurate quantitative DNA analysis in microfluidic systems
is required for the integration of PCR with μTAS, thus we anticipate that our device would have promising potential for applications
in a wide range of research. 相似文献
46.
Peter Krumbiegel Irina Lehmann Albin Alfreider Gisela J. Fritz David Boeckler Ulrike Rolle-Kampczyk 《Isotopes in environmental and health studies》2013,49(1):75-80
Background.?Studies conducted in Europe as well as in North and South America have tried to link Helicobacter pylori colonization with the drinking water supply, especially since H. pylori is known to survive quite well in water. Methods.?In 2000, a cohort of 1884 grade-two children from two rural counties surrounding the city of Leipzig, Germany (77.4% of the 1991/1992 birth cohort) were tested for H. pylori colonization using the [13C]urea breath test. A parent-completed questionnaire elicited details on living conditions and lifestyle habits including questions on the children's drinking water from sources other than public water supplies, swimming in natural waters, etc. In a second independent study, samples of well water, taken from 157 private wells still used in the two counties, were being tested for the presence of H. pylori, using polymerase chain reaction (PCR) method to determine relevant target DNA fragments of H. pylori. Results.?In county I, 5.7% of the children and in county II 6.6% tested H. pylori-positive. Cluster analyses of the questionnaire data in both counties pointed to ‘drinking water from other than municipal sources’, as the closest H. pylori-associated cluster variable. The cluster estimations were supported by odds ratio (OR) calculations with an OR?=?16.4 (95% confidence interval (CI) 3.1,…,88.5) for county I and OR?=?4.0 (95% CI 1.3,…,12.4) for county II. The PCR analyses showed H. pylori DNA fragments in 10.8% of the wells in county I and 9.2% in county II. The detection limit was set at 10 DNA copies corresponding to 125?bacteria/L, the average infestation of these wells was 931?bacteria/L. Conclusion.?Despite the fact that the microbiological and epidemiological data do not correspond except that both studies were conducted in the same geographical areas, the independent findings of H. pylori in well water in the same general areas where children do seem to drink water other than from the public water supply suggests that water may be an important source of H. pylori infection. 相似文献
47.
A novel projection modeling method for quantitative structure activity relationship (QSAR) and quantitative structure property relationship (QSPR) is developed in this paper. Orthogonalization of block variables is introduced to deal with the problem of variable selection. Projections based on least squares are used to construct the modeling space in order to search for the best regression directions for chemical modeling. A suitable prediction space for such a model is further defined to confine the usage range of the model. Three real data sets were analyzed to check the performance of the proposed modeling method. The results obtained from Monte‐Carlo cross‐validation (MCCV) showed that the proposed modeling method might provide better results for QSAR and QSPR modeling than PCR and PLS with respect to both fitting and prediction abilities. Copyright © 2007 John Wiley & Sons, Ltd. 相似文献
48.
Rui Tong Lijuan Zhang Qi Song Chuandeng Hu Xuee Chen Kai Lou Xiuqing Gong Yibo Gao Weijia Wen 《Electrophoresis》2019,40(12-13):1699-1707
Point‐of‐care detection for pathogen is of critical need for wide epidemic warning and medical diagnosis. In this work, we have designed and developed a fully portable and integrated microchip based real‐time polymerase chain reaction machine for rapid pathogen detection. The instrument consists of three functional components including heating, optical, and electrical modules, which are integrated into a portable compact box. The microchip is consumable material replaceable to meet various detection needs. Consequently, we demonstrated the outstanding performance of this portable machine for rapid detection of Salmonella and Escherichia coli O157:H7 with the advantage of time‐saving (~25 min), less samples consumption, portability, and user‐friendly operation. 相似文献
49.
Huan Yang Ying Zhou Pingtian Yu Yaya Yang Zhaoqun Jiao James P. Tam 《Natural product research》2019,33(9):1251-1256
A novel PCR technology was developed to detect short DNA fragments using species-specific primers for rapid and non-sequencing authentication of Bombyx batryticatus based on differences in the mitochondrial genome. Three specifically designed primer reactions were established to target species for the reliable identification of their commercial products. They were confirmed to have a high inter-species specificity and intra-species stability. The limit of detection was estimated as 1 ng of genomes for Beauveria bassiana and 100 pg for Bombyx mori and Metarhizium anisopliae. Furthermore, validation results demonstrated that raw materials and their processed products can be conveniently authenticated with good sensitivity and precision using this newly proposed approach. In particular, when counterfeits were assayed, these primer sets performed well, whereas COI barcoding technology did not. These could also assist in the discrimination and identification of adulterates of other animal-derived medicines in their pulverized and processed forms and even in complexes. 相似文献
50.
Akbari A Marthinsen G Lifjeld JT Albregtsen F Wennerberg L Stenseth NC Jakobsen KS 《Electrophoresis》2008,29(6):1273-1285
In CE the charged DNA strands are fractionated according to fragment lengths as they migrate through the capillary, since shorter DNA fragments pass through the sieving matrix faster. Multiplexed internal size standards are used to estimate the size of unknown DNA fragments. In the literature there are statements about migration abnormalities for the 250 and 340 bp fragments in the GeneScan-500 (GS500) internal size standards. Such anomalous migration of size standards could obviously introduce errors in the estimation of unknown fragments. Therefore, a number of analysis programs simply exclude some of these fragments. In the present work we first evaluate the effect of excluding each of the fragments in the internal size standards used in CE. Next, a method which is based on estimating the true values of the anomalous fragments is presented. The results obtained by the new method indicate a significant improvement compared to results obtained when one (or both) of the anomalous fragments in GS500 is (are) excluded or included when estimating the size of unknown DNA fragments. In the higher-molecular-weight region, the average error is reduced from 1.91 bp in ABI GeneMapper (excluding 250 bp) to 0.15 bp in the new method (using the estimated values for 250 and 340 bp). In the lower-molecular-weight region, excluding both fragments will improve the results by an average of 0.74 bp compared to ABI GeneMapper. 相似文献