共查询到20条相似文献,搜索用时 31 毫秒
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Kawachi R Koike Y Watanabe Y Nishio T Sakuda S Nagasawa H Oku T 《Molecular biotechnology》2004,27(3):179-186
This article describes a simple method for accurate rapid amplification of complementary deoxyribonucleic acid (cDNA) ends
(RACE), the distinctive feature being that only a gene-specific primer is used, without an anchor or adapter primer. Under
these conditions, Thermus aquaticus (Taq) polymerase synthesizes cDNA ends exactly, so that amplified products obtain a characteristic structure: a terminal inverted
repeat composed of a gene-specific primer and occasionally several nucleotides from its 3′ flanking sequence. These structures
suggest a hypothetical mechanism of cDNA end synthesis in which Taq DNA polymerase synthesizes a sequence complementary to the gene-specific primer at the 3′ end of the daughter strand by switching
the template to the 5′ terminal region through circularization of the DNA. As a result, the targeted cDNA will be efficiently
amplified with only a single gene-specific primer. This technique, which provides highly specific amplification of the 5′
and 3′ ends of a cDNA, is especially useful for isolation of cDNA when the corresponding messenger ribonucleic acid is scarce. 相似文献
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Yanwu Guo Lanqing Ma Yunpeng Ji Gaobin Pu Benye Liu Zhigao Du Guofeng Li Hechun Ye Hong Wang 《Molecular biotechnology》2011,47(2):152-156
We report a rapid and simple method for isolating the 5′-end of plant genes from genomic DNA by polymerase chain reaction
(PCR) with TATA-box-based degenerate primers (TDPs). The TDPs were specially designed according to the TATA box, which is
conserved in the promoter region of most plant genes. The unknown 5′-ends of several genes in different plants were isolated
by PCR with gene-specific primers of the known core fragment and the TDPs. Our method does not require the arduous RNA manipulations
and expensive enzyme treatments of the popular rapid amplification of cDNA ends (RACE) and its variants, and so could be a
cheap practical alternative. 相似文献
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TheenvelopeproteinofhepatitisBvirus(HBV)consistsofthreeproteins:small(S),middle(M)andlarge(L)[1].TheSproteincarriesalltheinformationrequiredforcellularlipidsmobilization,subviralparticleformationandsecretion.Ithasbeensuccessfullydevelopedasacarriertoexpressf… 相似文献
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Sequence-specific binding of the influenza virus RNA polymerase to sequences located at the 5' ends of the viral RNAs. 总被引:9,自引:7,他引:2
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The enzymatic activity of recombinant influenza virus RNA polymerase is strictly dependent on the addition of a template RNA containing 5' and 3' viral sequences. Here we report the analysis of the binding specificity and physical characterization of the complex by using gel shift, modification interference, and density gradient techniques. The 13S complex binds specifically to short synthetic RNAs that mimic the partially double stranded panhandle structures found at the termini of both viral RNA and cRNA. The polymerase will also bind independently to the single-stranded 5' or 3' ends of viral RNA. It binds most strongly to specific sequences within the 5' end but is unable to bind these sequences in the context of a completely double stranded structure. Modification interference analysis identified the short sequence motifs at the 5' ends of the viral RNA and cRNA templates that are critical for binding. 相似文献
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Interleukin-5 (IL-5) is thought to be a key cytokine in allergic inflammation. Pig IL-5 was cloned, sequenced, and expressed
to enable us to study of the biological role of IL-5 in pigs used in a model for allergen-induced late-phase reactions. These
pigs were sensitized to proteins extracted from Ascaris suum, resulting in hypersensitivity to this antigen in both the skin and airways, and a slight blood eosinophilia. Peripheral
blood mononuclear cells from antigen-sensitized pigs were isolated and polyclonally stimulated. Total RNA was extracted and
reverse transcribed into cDNA. IL-5 primers based on the cow IL-5 cDNA sequence were used to obtain an initial polymerase
chain reaction product. 3′ rapid amplification of cDNA ends (3′RACE) and 5′RACE procedures were applied to identify the 3′
and 5′ ends, respectively. The full-length pig IL-5 cDNA is 405 base pairs long. Mature pig IL-5 was expressed in Escherichia coli with a His-tag for purification. The IL-5 protein is 115 amino acids long, has an estimated molecular weight of 14 000 M
r
and forms a biologically active homodimer of 28 000 M
r
. Pig IL-5 shows 65% amino acid identity to the human IL-5 sequence and 90, 88, 83, 62, and 61% identity to the cow, sheep,
horse, mouse, and rat counterparts.
Received: 29 June 1999 / Revised: 22 September 1999 相似文献
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Summary The finding that ribozymes can catalyze RNA chain elongation has led to the proposal that an early self-replicating system
could have consisted of RNA alone. In such a chain elongation reaction, theTetrahymena ribozyme was found to select 3′, 5′-linked substrates from a pool that contained a large molar excess of 2′, 5′-linked dinucleotides.
The enzyme neither reacted with nor was inhibited by 2′, 5′ phosphodiester linkages. The ability to exclude incorrectly linked
substrates would have conferred an important selective advantage to a primordial RNA molecule with RNA replicase activity. 相似文献
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Hilario E 《Molecular biotechnology》2004,28(1):77-80
There are two ways to label a DNA molecular; by the ends or all along the molecule. End labeling can be performed at the 3′-
or 5′-end. Labeling at the 3′ end is performed by filling 3′-end recessed ends with a mixture or labeled and unlabeled dNTPs
using Klenow or T4 DNA polymerases. Both reactions are template dependent. Terminal deoxynucleotide transferase incorporates
dNTPs at the 3′ end of any kind of DNA molecule or RNA. Labels incorporated at the 3′-end of the DNA molecule prevent any
further extension or ligation to any other molecule, but this can be overcome by labeling the 5′-end of the desired DNA molecule.
5′-end labeling is performed by enzymatic methods (T4 polynucleotide kinase exchange and forward reactions), by chemical modification
of sensitized oligonucleotides with phosphoroamidite, or by combined methods. Probe cleanup is recommended when high background
problems occur, but caution should be taken not to damage the attached probe with harsh chemicals or by light exposure. 相似文献
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