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11.
Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction 总被引:83,自引:0,他引:83
Gene Splicing by Overlap Extension or "gene SOEing" is a PCR-based method of recombining DNA sequences without reliance on restriction sites and of directly generating mutated DNA fragments in vitro. By modifying the sequences incorporated into the 5'-ends of the primers, any pair of polymerase chain reaction products can be made to share a common sequence at one end. Under polymerase chain reaction conditions, the common sequence allows strands from two different fragments to hybridize to one another, forming an overlap. Extension of this overlap by DNA polymerase yields a recombinant molecule. This powerful and technically simple approach offers many advantages over conventional approaches for manipulating gene sequences. 相似文献
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Iris Maldener Wolfgang Lockau Yuping Cai C. Peter Wolk 《Molecular & general genetics : MGG》1991,225(1):113-120
Summary It has been suggested that a calcium-dependent intracellular protease of the cyanobacterium, Anabaena sp., participates in the differentiation of heterocysts, cells that are specialized for fixation of N2. Clones of the structural gene (designated prcA) for this protease from Anabaena variabilis strain ATCC 29413 and Anabaena sp. strain PCC 7120 were identified via their expression in Escherichia coli. The prcA gene from A. variabilis was sequenced. The genes of both strains, mutated by insertion of a drug resistance cassette, were returned to these same strains of Anabaena on suicide plasmids. The method of sacB-mediated positive selection for double recombinants was used to achieve replacement of the wild-type prcA genes by the mutated forms. The resulting mutants, which lacked Ca2+-dependent protease activity, were not impaired in heterocyst formation and grew on N2 as sole nitrogen source. 相似文献
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Chlorinated methanes are important environmental pollutants, which can be metabolized by bacteria. The biotransformation of chlorinated methanes by bacteria has been shown to be due either to gratuitous metabolism (cometabolism) or their use as a source of carbon and energy. The reactions which result in carbon-halogen bond cleavage include substitutive, reductive, oxygenative, and gem-elimination mechanisms. Certain methylotrophic bacteria can use dichloromethane as a source of carbon and energy. Dichloromethane dehalogenase catalyzes the first substitutive reaction in this metabolism. The enzyme shows a 1010-fold rate enhancement over the reaction of the bisulfide anion with dichloromethane in water. Pseudomonas putida G786 synthesizes cytochrome P-450CAM which catalyzes the gratuitous reduction of chlorinated methanes. These studies with purified enzymes are beginning to reveal more detailed mechanistic features of bacterial chlorinated methane metabolism.Abbreviations DNA
deoxyribonucleic acid
- kcat
catalytic first order rate constant for an enzyme catalyzed reaction
- KM
Michaelis constant for an enzyme catalyzed reaction
- MNDO
modified neglect of diatomic overlap
- PIMA
pattern induced multialignment
- DCMD
dichloromethane dehalogenase 相似文献
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<正> 为了在TSr(Bgl Ⅱ-1)核苷酸顺序中寻找有无类似α—顺序的冷点区顺序,我们在微型电子计算机上编制了查找相似序列顺序(WSS程序)。本程序的特点是运行速度快,无重复扫描,可自行选择欲查找的相似百分比和百分比精度,它不仅适用于寻找内切酶的酶切点,而且可以在相当长度的已测序DNA顺序中快速准确地检出碱基位置和数量发生随机变异的DNA相似片段,并直接计算、打印出相似百分比值。 相似文献
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Localization of a beta-crystallin gene, Hu beta A3/A1 (gene symbol: CRYB1), to the long arm of human chromosome 17 总被引:3,自引:0,他引:3
M L Law G Y Cai J Hartz F T Kao D Hogg M L Breitman L C Tsui 《Cytogenetics and cell genetics》1986,42(4):202-207
We have assigned a human beta-crystallin gene, Hu beta A3/A1 (gene symbol: CRYB1), to chromosome 17 using a panel of 19 human-hamster somatic cell hybrids and blot-hybridization analysis of cell hybrid DNA. Positive probe-hybridization signal was detected in a hybrid that had lost the short arm of human chromosome 17 but retained the long arm, translocated to a hamster chromosome. In addition, in situ hybridization analysis of metaphase chromosome spreads of this cell line suggested that the most probable location for CRYB1 is on the long arm of chromosome 17, in the region q21. 相似文献