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Here, we describe the application of a RecA-based cloning technology to generate full-length cDNA libraries enriched for genes that are differentially expressed between tumor and normal tissue samples. First, we show that the RecA-based method can be used to enrich cDNA libraries for several target genes in a single reaction. Then, we demonstrate that this method can be extended to enrich a cDNA library for many full-length cDNA clones using fragments derived from a subtracted cDNA population. The results of these studies show that this RecA-mediated cloning technology can be used to convert subtracted cDNAs or a mixture of several cDNA fragments corresponding to differentially expressed genes into a full-length library in a single reaction. This procedure yields a population of expression-ready clones that can be used for further high-throughput functional screening. 相似文献
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Generation of Arabidopsis Mutants by Heterologous Expression of a Full-Length cDNA Library from Tomato Fruits 总被引:1,自引:0,他引:1
Yi-Hong Wang Chris M. Mosebach Abraham S. Kibbey Marcie K. Ryhal Angelica D. Jones Julie A. Palmer Leon V. Kochian 《Plant Molecular Biology Reporter》2009,27(4):454-461
Heterologous expression of cDNA library in Arabidopsis and other plants has been used for gene identifications. To identify
functions of tomato genes, we expressed a tomato full-length cDNA library in Arabidopsis thaliana and generated over 7,000 mutants. We constructed a tomato cDNA library with a plant transformation-ready binary vector that
contained a higher percentage of full-length cDNAs since synthesized double-stranded cDNA was size-selected using gel electrophoresis,
with cDNA sizes of 2–5 kb being gel-purified for ligation onto the binary vector. Sequencing of 81 cDNA clones indicates that
75% (61) are full-length genes, which is similar to sequencing of inserted cDNA in Arabidopsis. The library was used to transform
Arabidopsis plants. Among the 7,000 mutants, one was found to be a dwarf due to the expression of an ATP synthase, and another
vegetative mutant did not produce flowers even after 7 months. The technique was validated by reintroducing the tomato ribosomal
protein L9 gene and can be used in any other plant species as a gene discovery tool. 相似文献
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Zhulidov PA Bogdanova EA Shcheglov AS Shagina IA Vagner LL Khazpekov GL Kozhemiako VV Luk'ianov SA Shagin DA 《Bioorganicheskaia khimiia》2005,31(2):186-194
We developed a new method for the preparation of normalized cDNA libraries enriched with full-length sequences. It is based on the properties of the recently characterized duplex-specific nuclease from the hepatopancreas of the Kamchatka crab. The duplex-specific nuclease is thermostable, it effectively cleaves double-stranded DNA and is inactive toward single-stranded DNA (Shagin et al., Genome Res., 2002, vol. 12, pp. 1935-1942). Our method enables the normalization of cDNA samples enriched with full-length sequences without use of laborious and ineffective stages of physical separation. The efficiency of the method was demonstrated in model experiments using cDNA samples from several human tissues. 相似文献
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cDNA文库与RACE方法结合克隆一个马铃薯病程相关蛋白基因cDNA 总被引:5,自引:0,他引:5
为克隆马铃薯晚疫病抗性相关基因,深入研究马铃薯晚疫病抗性机制,应用SMART LD—PCR技术,以晚疫病菌混合小种诱导48h的水平抗性马铃薯(Solanum tuberosum L.)(R—gene—free)叶片为材料,构建了一个富集晚疫病抗性相关基因的cDNA文库。为提高克隆全长cDNA的效率,将cDNA文库与RACE技术结合,依据本实验室得到的病原诱导表达片段测序结果,在其内部设计两个特异引物,与文库载体臂上的通用引物配对,以文库噬菌体DNA为模板,用高保真PCR分别扩增出cDNA5′端与3′端,从而简便、快捷地得到全长cDNA序列。采用此方法,在马铃薯中克隆了一个受晚疫病菌诱导表达的cDNA,该cDNA长904bp,5′端有29bp的非翻译区,3′端具有完整的polyA尾,包含一个678bp的完整开放阅读框架,编码226个氨基酸(GenBank登录号:AY 185207)。BLAST检索发现其氨基酸序列与烟草一个新的病程相关蛋白基因NtPRp27具有90%的同源性,在马铃薯中尚未发现与之同源的已知基因。Northern杂交结果表明,水杨酸(SA)、茉莉酸(JA)、茉莉酸甲酯(MeJA)、机械伤害和渗透胁迫都能诱导该基因表达。该基因可能是马铃薯一个新的病程相关蛋白基因。 相似文献
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P.?A.?Zhulidov E.?A.?Bogdanova A.?S.?Shcheglov I.?A.?Shagina L.?L.?Wagner G.?L.?Khazpekov V.?V.?Kozhemyako S.?A.?Lukyanov
We developed a new method for the preparation of normalized cDNA libraries enriched with full-length sequences. It is based on the properties of the recently characterized duplex-specific nuclease from the hepatopancreas of the Kamchatka crab. The duplex-specific nuclease is thermostable, effectively cleaves double-stranded DNA, and is inactive toward single-stranded DNA (Shagin et al., Genome Res., 2002, vol. 12, pp. 1935–1942). Our method enables the normalization of cDNA samples enriched with full-length sequences without use of laborious and ineffective stages of physical separation. The efficiency of the method was demonstrated in model experiments using cDNA samples from several human tissues.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 186–194.Original Russian Text Copyright © 2005 by Zhulidov, Bogdanova, Shcheglov, Shagina, Wagner, Khazpekov, Kozhemyako, Lukyanov, Shagin. 相似文献
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针对细菌mRNA poly(A)化位点的高度多态性,利用oligo(dT)与poly(A)特异结合的特性,以oligo(dT)一纤维素纯化mRNA,并以oligo(dT)18为引物逆转录合成cDNA,用限制性内切酶消化cDNA,所得的限制性内切酶片段与通用接头相连,通过10个选择性引物组合进行选择性PCR,使各片段得以扩增并分布于10个亚组中,并进行克隆,成功地克隆了100多个基因片段,已对其中40个进行了测序分析,探讨了限制性显示PCR技术在细菌poly(A)化mRNA cDNA库构建中的应用价值。 相似文献
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PCR-enriched cDNA pool method for cloning of gene homologues 总被引:3,自引:0,他引:3
Minglin Lang Yuxiu Zhang Ziqiu Guan Tuanyao Chai 《Plant Molecular Biology Reporter》2005,23(3):219-226
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Construction of a human full-length cDNA bank 总被引:14,自引:0,他引:14
Seishi Kato Shingo Sekine Su-Wan Oh Nam-Soon Kim Yuri Umezawa Naoto Abe Midori Yokoyama-Kobayashi Takashi Aoki 《Gene》1994,150(2):243-250
We aimed to construct a full-length cDNA bank from an entire set of human genes and to analyze the function of a protein encoded by each cDNA. To achieve this purpose, a multifunctional phagemid shuttle vector, pKAl, was constructed for preparing a high-quality cDNA library composed of full-length cDNA clones which can be sequenced and expressed in vitro and in mammalian cells without subcloning the cDNA fragment into other vectors. Using this as a vector primer, we have prepared a prototype of the bank composed of full-length cDNAs encoding 236 human proteins whose amino acid sequences are identical or similar to known proteins. Most cDNAs contain a putative cap site sequence, some of which show a pyrimidine-rich conserved sequence exhibiting polymorphism. It was confirmed that the vector permits efficient in vitro translation, expression in mammalian cells and the preparation of nested deletion mutants. 相似文献
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The ability to generate and obtain full-length (FL) cDNAs is of critical importance to the field of genomics. Most cDNAs in a traditional cDNA library lack the initiating 5' ATG, making it difficult to obtain a FL clone. We report here on an improved protocol for the preparation of FL enriched cDNA libraries. We demonstrate that if good quality RNA is used in the cDNA synthesis, high-quality, FL cDNA can be generated for messages upward of 7 kb. In addition, we demonstrate the utility of size fractionation as a means to produce libraries containing a high percentage of initiating 5' ATG containing clones with insert sizes greater than 4 kb. The method is simple, cost efficient, and can be performed in most laboratories equipped to perform molecular biology. Lastly, the novel methodologies used in the analysis of the cDNA and library should prove useful to others working to create high-quality cDNA libraries. 相似文献
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Identification of unique transcripts from a mouse full-length, subtracted inner ear cDNA library 总被引:8,自引:0,他引:8
Beisel KW Shiraki T Morris KA Pompeia C Kachar B Arakawa T Bono H Kawai J Hayashizaki Y Carninci P 《Genomics》2004,83(6):34699-1023
A small-scale full-length library construction approach was developed to facilitate production of a mouse full-length cDNA encyclopedia representing approximately 250 enriched, normalized, and/or subtracted cDNA libraries. One library produced using this approach was a subtracted adult mouse inner ear cDNA library (sIEa). The average size of the inserts was approximately 2.5 kb, with the majority ranging from 0.5 to 7.0 kb. From this library 22,574 sequence reads were obtained from 15,958 independent clones. Sequencing and chromosomal localization established 5240 clusters, with 1302 clusters being unique and 359 representing new ESTs. Our sIEa library contributed 56.1% of the 7773 nonredundant Unigene clusters associated with the four mouse inner ear libraries in the NCBI dbEST. Based on homologous chromosomal regions between human and mouse, we identified 1018 UniGene clusters associated with the deafness locus critical regions. Of these, 59 clusters were found only in our sIEa library and represented approximately 50% of the identified critical regions. 相似文献
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Library subtraction of in vitro cDNA libraries to identify differentially expressed genes in scrapie infection. 总被引:26,自引:4,他引:22
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We have developed a system where double-stranded cDNA can be amplified using a synthetic oligonucleotide primer and the polymerase chain reaction, generating cDNA libraries in vitro. Using a library subtraction strategy (1), scrapie and control brain in vitro cDNA libraries were used to identify sequences whose expression is modulated in scrapie infection. One of these sequences represents beta-2 microglobulin, while the other two have not been previously described. The use of in vitro libraries offers increased speed and efficiency of construction, and their subtraction is more efficient and powerful, compared with the previous system (1). 相似文献