共查询到19条相似文献,搜索用时 93 毫秒
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使用与Gateway技术兼容的T载体获得入门克隆 总被引:8,自引:0,他引:8
与Gateway技术兼容的农杆菌双元载体系统已开始应用于植物功能基因组的研究,但应用这些载体系统的一个瓶颈问题,是如何简单、经济和高效地将PCR产物或其他来源的目的DNA片段构建到入门载体上获得入门克隆.为此,将传统的TA克隆技术与Gateway重组克隆技术进行整合,构建了与Gateway技术兼容的两种TA克隆载体,用于在克隆PCR产物或其他来源的目的DNA片段的同时获得入门克隆.利用兼容Gateway技术的TA克隆载体有效地解决了上述瓶颈问题. 相似文献
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DNA片段的亚克隆通常包括连接、转化、筛选、鉴定等步骤。 一、载体的选择 根据待亚克隆的DNA片段的两端顺序、用途(如准备作DNA顺序分析、大量扩增或作酶切图谱分析等)和载体DNA分子带有的限制酶位置,选定合适的载体。 相似文献
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操作含长插入片段的DNA克隆时 ,经常需要进行亚克隆和测序实验。通常的方法首先是得到插入片段的限制性内切酶谱 ,然后选择合适的内切酶消化DNA ,分离靶片段 ,将其连接入质粒载体中进行下一步操作。但这种方法工作量大 ,步骤繁琐。在此 ,介绍一种不需要做限制性内切酶谱分析 ,而根据靶片段的旁侧序列直接进行亚克隆实验的方法。首先 ,选择合适的限制性内切酶消化含长插入片段的DNA克隆 ,其中一种酶切在已知的旁侧序列上 ,另一为随机选择 ;然后酶切混合物与线性化的质粒载体连接 ,转化细菌得到一“亚克隆库” ;将其中的克隆挑选入 96孔板培养后 ,按行或列混合菌液得到相应的“pool” ;最后 ,用PCR方法筛选获得含靶DNA片段的阳性克隆 ,其中所用的引物一个与已知的旁侧DNA序列配对 ,另一个与质粒载体上序列配对 ,PCR扩增已知的旁侧DNA片段以鉴定阳性克隆。多次独立实验表明该方法简单有效 ,可广泛用于亚克隆和DNA步移实验 相似文献
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近年来,一种用于DNA克隆的双载体系统发展十分迅速。这种系统有许多优点,特别是它广谱寄主的特性,是我们过去常用的一些载体系统所不具有的。在实际应用中,它已显示出作为一种有效的多用途的DNA克隆载体的潜力。这就是以PRK290质粒为克隆载体、以PRK2013质粒为帮助质粒(helper Plasmid)可转移进各种革兰氏阴性菌的双载系统。 相似文献
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DNA分子克隆是基本的分子生物学实验技术,传统的分子克隆方法大多需经过酶切链接过程,但在某些情况下,没有合适的酶切位点往往会成为阻碍克隆进行的障碍.本文描述了一种新的分子克隆方法,称为不依赖酶切和链接的分子克隆(RLIC).利用RLIC,将3种不同大小的DNA片段克隆到3种不同载体,证明了这种方法的有效性和可靠性.由于该方法不受限制性酶切序列限制,省去了酶切连接步骤,因此具有很大的灵活性和简便性,在分子生物学研究方面有广泛应用前景. 相似文献
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鸡肠道微生物菌群经PCR-DGGE分析,回收PCR-DGGE分析胶上的一条DNA片段,回收的DNA片段再重复进行2次PCR-DGGE分析,以及分别用PCR反复循环扩增和PCR高保真酶扩增后再进行DGGE分析等方法研究PCR-DGGE分析中多条带产生原因。结果显示PCR-DGGE分析中多条带产生原因可能是作PCR扩增模板的DNA混杂有少量其他DNA片段,多条带现象不易被消除。DGGE分析胶上的DNA片段测序时,将该DNA片段回收、PCR扩增后克隆,提取多个阳性克隆菌的质粒DNA片段,分别与其原目的DNA片段进行DGGE分析,在DGGE分析胶上选取与原目的DNA片段处于同一电泳位置的质粒DNA测序,提高测序的准确性。 相似文献
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This paper describes the construction of 'Prime' cloning vectors, which include phage lambda and plasmid vectors useful for functional cloning in oocytes, yeast, and mammalian cells, and their use in a 'Prime' cloning system. The system takes advantage of the very active and precise 3' exonuclease activity of T4 DNA polymerase to produce single-stranded (ss) ends (cut-back) of vector and insert DNA. This results in the highly efficient directional cloning of cDNA and PCR-amplified DNA. The system obviates the need to digest insert DNA with a restriction endonuclease to unveil cloning sites, and thus eliminates the chance of internal digestion of the insert DNA. The cloning of PCR-amplified DNA, which is sometimes difficult, is made routine with this system. The 'Prime' sequence is included in vector cloning sites and cDNA and PCR primers. The 'Prime' sequence was chosen so that the ss sticky ends are nonpalindromic and will hybridize only to the appropriate partners. This makes cloning with the 'Prime' system very efficient, because neither the vector nor insert DNA is lost to unproductive self-hybridization. 相似文献
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基因组序列的功能分析以及代谢途径的构建改造等都需要克隆目的DNA。获得大片段DNA序列的方法有构建和筛选基因文库,PCR扩增,体外大片段DNA合成和组装等,但体内重组直接克隆的方法在操作、克隆长片段和应用等方面更具优势。介绍了Red/ET重组介导的大片段DNA体内直接克隆的主要方法及其应用。 相似文献
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Generation of a 50,000-member human DNA library with an average DNA insert size of 75-100 kbp in a bacteriophage P1 cloning vector 总被引:3,自引:0,他引:3
A bacteriophage P1 cloning system that permits the isolation and amplification of cloned DNA fragments as large as 100 kbp was described previously. We have now utilized a similar system to generate a 50,000-member human DNA library with DNA inserts ranging in size from 75 to 100 kbp. Two major obstacles were overcome in constructing the library. The first concerned the mcrAB restriction system of Escherichia coli, which degrades DNA containing MeC and interferes with the recovery of cloned human DNA inserts. In the P1 cloning system, the effect of the Mcr restriction activity is to decrease recovery of cloned inserts by about 35-fold when the activity is in the host cell line and by about 3-fold when the activity is in the cells used to prepare the packaging extract. To circumvent this problem we inactivated, by mutation, the McrAB proteins in both components of the cloning system. The second obstacle concerned the preferential cloning of small DNA fragments from a population of fragments ranging in size from 20 to 100 kbp. To deal with this problem we first modified the P1 lysogen used to prepare the in vitro head-tail packaging extract so that it would produce 12 times as many large P1 heads (head capacity about 110 kbp) as small P1 heads (head capacity about 45 kbp). We then restructured the P1 cloning vector so that it could be used to produce vector "arm" fragments that could be ligated to insert DNA at only one end. This prevented the formation of long concatamers consisting of alternating units of vector and insert DNA and prohibited the packaging of small inserts in large phage heads. Using the insert-biased large head extract, the arms vector, and size-selected human DNA fragments, we showed that as much as 90% of recovered transformants contained inserts in the desired high molecular weight range. 相似文献
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Human telomeres have been successfully cloned in Saccharomyces cerevisiae by complementing deficient yeast artificial chromosomes (YACs). This technique allows cloning of DNA sequences that can recognize particular chromosomal ends, and therefore facilitates the mapping of eukaryotic genomes. Although the biology of adopting foreign telomeres in yeast is not fully understood, the cloning system itself seems to be a useful tool for constructing telomeric DNA libraries from higher eukaryotes. Here we describe the techniques that are currently being used in cloning of telomeric DNA. 相似文献
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Direct cloning of a long continuous genome segment in a Bacillus subtilis genome vector was demonstrated for the first time. Two small DNA fragments had to be installed in the vector prior to cloning. The DNA between these two fragments was cloned via homologous recombination. The efficiency of cloning was estimated using the 3,573-kb genome of a cyanobacterium, Synechocystis sp. PCC 6803. Recombinants were selected using the internal selection system of the Bacillus genome vector or with the antibiotic resistance marker in the cyanobacterial genome. Designated genomic segments as large as 77-kb were cloned by means of a single procedure. Cloning efficiency is affected by the molecular weight of the donor DNA and the size of the DNA to be cloned. The method is suitable for direct target cloning of large-sized DNA. 相似文献
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《Gene Analysis Techniques》1990,7(5):119-125
Human telomeres have been succesfully cloned in Saccharomyces cerevisiae by complementing deficient yeast artificial chromosomes (YACs). This technique allows cloning of DNA sequences that can recognize particular chromosomal ends, and therefore facilitates the mapping of eukaryotic genomes. Although the biology of adopting foreign telomeres in yeast is not fully understood, the cloning system itself seems to be a useful tool for constructing telomeric DNA libraries from higher eukaryotes. Here we describe the techniques that are currently being used in cloning of telomeric DNA. 相似文献
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A Versatile Zero Background T-Vector System for Gene Cloning and Functional Genomics 总被引:4,自引:0,他引:4 下载免费PDF全文
Songbiao Chen Pattavipha Songkumarn Jianli Liu Guo-Liang Wang 《Plant physiology》2009,150(3):1111-1121
With the recent availability of complete genomic sequences of many organisms, high-throughput and cost-efficient systems for gene cloning and functional analysis are in great demand. Although site-specific recombination-based cloning systems, such as Gateway cloning technology, are extremely useful for efficient transfer of DNA fragments into multiple destination vectors, the two-step cloning process is time consuming and expensive. Here, we report a zero background TA cloning system that provides simple and high-efficiency direct cloning of PCR-amplified DNA fragments with almost no self-ligation. The improved T-vector system takes advantage of the restriction enzyme XcmI to generate a T-overhang after digestion and the negative selection marker gene ccdB to eliminate the self-ligation background after transformation. We demonstrate the feasibility and flexibility of the technology by developing a set of transient and stable transformation vectors for constitutive gene expression, gene silencing, protein tagging, protein subcellular localization detection, and promoter fragment activity analysis in plants. Because the system can be easily adapted for developing specialized expression vectors for other organisms, zero background TA provides a general, cost-efficient, and high-throughput platform that complements the Gateway cloning system for gene cloning and functional genomics. 相似文献
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We have developed a novel way to use the Cre/loxP system for in vitro manipulation of DNA and a technique to clone DNA into circular episomes. The method is fast, reliable, and allowsflexible cloning of DNA fragments into episomes containing a loxP site. We show that a loxP site can serve as a universal target site to clone a DNA fragment digested with any restriction enzyme(s). This technique abolishes the need for compatible restriction sites in cloning vectors and targets by generating custom-designed 5' 3', or blunt ends in the desired orientation and reading frame in the vector Therefore, this method eliminates the limitations encountered when DNA fragments are cloned into vectors with a confined number of cloning sites. The 34-bp loxP sequence assures uniqueness, even when large episomes are manipulated. We present three examples, including the manipulation of a bacterial artificial chromosome. Because DNA manipulation takes place at a loxP site, we refer to this technique as loxP-directed cloning. 相似文献