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一种用于构建红色红曲霉基因组Cosmid文库的大片段DNA制备方法 总被引:1,自引:0,他引:1
摘要:【目的】制备用于构建红色红曲霉cosmid文库的大片段基因组DNA。【方法】采用优化的酚氯仿抽提法制备DNA,并利用Sau3AI切割至平均大小为40 kb,然后使用Stratagene包装蛋白构建cosmid文库。基于PCR法使用同源探针从该文库中进行了目的基因的筛选。【结果】制备了浓度为5 μg/μL,平均片段大小大于48 kb的红色红曲霉大片段基因组DNA。利用该DNA构建的cosmid文库基因组覆盖倍数为10,并筛选到了含有目的片段的cosmid。【结论】通过该方法制备红色红曲霉大片段基因组D 相似文献
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介绍了构建大腹圆蛛Fosmid基因组文库及牵引丝蛋白(MaSp)基因克隆筛选的全过程.采用改良CTAB法提取大片段基因组DNA,通过自主构建的电洗脱核酸回收装置分离回收30~40kbDNA片段,经补平磷酸化、与pCC2FOS载体连接、体外包装和转染EPI300TM-T1R,首次构建了无偏向性的大腹圆蛛Fosmid文库,其滴度为4.5×105cfu/mL,覆盖基因组倍数为10.以α-32P标记寡核苷酸探针对文库进行初步筛选,获得含MaSp基因的12个阳性克隆.该文库符合Fosmid文库的品质要求,为进一步筛选并研究大腹圆蛛MaSp基因序列奠定了基础. 相似文献
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为了研究蛋白质之间的相互作用,利用Gateway技术构建了镜鲤(Cyprinus carpio Linnaeus)骨骼肌酵母双杂交cDNA文库.经检测表明,构建的初级cDNA文库的库容量为8×106CFU;酵母双杂交cDNA文库的库容量为6.64×106CFU,插入片段集中在1-2 kb之间,具有较好的多态性.随机挑取的24个克隆没有空载体,全部发生了重组.较高的库容量、较长的插入片段以及较高的重组率保证了文库的完整性和覆盖度.镜鲤骨骼肌酵母双杂交cDNA文库的构建为克隆全长目的基因及研究影响骨骼肌发育的信号传导通路奠定了基础. 相似文献
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目的:对4个消减cDNA文库中筛选到的特异基因片段进行功能分析,为筛选津田芜菁和赤丸芜菁花青素合成的特异基因和代谢途径奠定基础。方法:以不同处理的津田芜菁和赤丸芜菁块根为材料,采用抑制削减杂交构建4个消减cDNA文库并富集特异基因群体,同时对消减文库的特异基因片段进行初步的生物信息学分析。结果:通过功能聚类、代谢途径分析和基因注释等手段分析了消减cDNA文库的特异基因片段。结论:对消减文库特异基因片段的功能分析,为进一步分离和鉴定依光型和非依光型花青素合成相关基因奠定了基础。 相似文献
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《基因组学与应用生物学》2017,(11)
RNA-seq是利用深度测序进行转录组分析的技术,并且在新一代测序的主流平台Illumina上运用广泛。针对Illumina平台的RNA-Seq建库,目前有多个不同类型的建库方法支持该技术,但是在操作步骤和成本上有很大差距。本研究分别试验了Tru Seq RNA文库制备、NEBNext RNA文库制备以及KAPA RNA文库制备3种方法,首次系统地对这3种建库方法得到的片段长度,浓度和建库流程进行了比较分析。研究显示,3种文库的片段长度都集中在300~1 000 bp之间,KAPA RNA文库制备得到的文库浓度最高,该制备方法只需要进行一次磁珠纯化,操作简便,成本低,所需要的起始量低,适用于进行大规模的建库实验。 相似文献
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目的:构建甜菜夜蛾触角全长cDNA文库。方法:利用TRIzol试剂提取甜菜夜蛾触角总RNA,以此为模板,通过SMAR-TScribeTM反转录酶反转录合成第一链cDNA,引物扩增获得双链cDNA,经Proteinase K消化、SfiⅠ酶切和CHROMA SPIN-400Column分级分离后,收集400~2 000 bp之间的片段重组于改造的pUC19载体并转化至大肠杆菌Escherichia coli DH5α,最终构建获得甜菜夜蛾触角全长cDNA文库。结果:对文库进行滴度测定和重组率分析,结果表明构建的cDNA初级文库滴度为1.6×107pfu/ml,重组率为94%,插入片段大小为0.5~3.0 kb,平均长度在1 kb以上,表明构建获得的文库是一个高质量的文库。结论:该文库的构建为今后克隆甜菜夜蛾嗅觉相关基因奠定了基础。 相似文献
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灰飞虱高带毒(RSV)群体酵母双杂交cDNA文库的构建 总被引:1,自引:0,他引:1
为了研究灰飞虱Laodelphax striatellus Fallén与水稻条纹病毒(rice stripe virus, RSV)互作机制, 本研究构建了灰飞虱高带毒群体酵母双杂交cDNA文库。以实验室筛选的灰飞虱高带毒群体为材料, 分离纯化mRNA, 反转录合成双链cDNA, 并连接三框型接头, 层析柱分级纯化。采用同源重组反应制备三框型cDNA入门文库, 再通过同源重组将入门文库转移到Gateway兼容载体pGADT7-DEST上, 构建获得酵母双杂交cDNA文库。检测结果表明: 文库库容量为3.68×107 cfu, 扩增文库滴度为2.62×1010 cfu/mL; 文库重组率大于95%, cDNA插入片段平均长度>1 kb, 达到了标准cDNA文库的要求。灰飞虱高带毒群体酵母双杂交cDNA文库的构建为开展昆虫介体与水稻条纹病毒互作机制的研究奠定了基础。 相似文献
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Construction and characterization of a bacterial artificial chromosome library of Sorghum bicolor. 总被引:30,自引:1,他引:29 下载免费PDF全文
The construction of representative large insert DNA libraries is critical for the analysis of complex genomes. The predominant vector system for such work is the yeast artificial chromosome (YAC) system. Despite the success of YACs, many problems have been described including: chimerism, tedious steps in library construction and low yields of YAC insert DNA. Recently a new E.coli based system has been developed, the bacterial artificial chromosome (BAC) system, which offers many potential advantages over YACs. We tested the BAC system in plants by constructing an ordered 13,440 clone sorghum BAC library. The library has a combined average insert size, from single and double size selections, of 157 kb. Sorghum inserts of up to 315 kb were isolated and shown to be stable when grown for over 100 generations in liquid media. No chimeric clones were detected as determined by fluorescence in situ hybridization of ten BAC clones to metaphase and interphase S.bicolor nuclei. The library was screened with six sorghum probes and three maize probes and all but one sorghum probe hybridized to at least one BAC clone in the library. To facilitate chromosome walking with the BAC system, methods were developed to isolate the proximal ends of restriction fragments inserted into the BAC vector and used to isolate both the left and right ends of six randomly selected BAC clones. These results demonstrate that the S. bicolor BAC library will be useful for several physical mapping and map-based cloning applications not only in sorghum but other related cereal genomes, such as maize. Furthermore, we conclude that the BAC system is suitable for most large genome applications, is more 'user friendly' than the YAC system, and will likely lead to rapid progress in cloning biologically significant genes from plants. 相似文献
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灰树花总DNA的制备及基因组文库的构建A 总被引:3,自引:0,他引:3
灰树花是一种珍贵的药用真菌,因为多糖含量较高,较难获得高质量的总DNA,本文提出了一种制备高质量灰树花总DNA及构建灰树花基因组文库的方法。该方法制备的灰树花总DNA,经Sau3AI酶切后,用于构建基因组文库,可得到2×105个转化子/50mg,平均插入片段为14kb。本研究为下一步克隆灰树花中的基因以及进行其他分子生物学研究奠定了基础。Abstract: Grifola frondosa, is a valuable medicinal fungus. High quality total genomic DNA is difficult to prepare due to its high polysaccharide content. A method for the preparation of Grifola frondosa total genomic DNA and construction of Grifola frondosa, genomic library is described. Genomic DNA prepared by this method is digested by Sau3A I restriction enzyme. Constructed genomic library give a titer of 2×105 transformants/50mg , with a average insert size of 14kb. This has paved way for the cloning of other Grifola frondosa genes and molecular biology studies. 相似文献
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Identification of recombinant phages containing sequences from different rat myosin heavy chain genes. 总被引:15,自引:3,他引:12 下载免费PDF全文
U Nudel D Katcoff Y Carmon D Zevin-Sonkin Z Levi Y Shaul M Shani D Yaffe 《Nucleic acids research》1980,8(10):2133-2146
The construction and identification of a recombinant plasmid containing a cDNA insert which hybridizes specifically to myosin heavy chain mRNA is described. The plasmid was used as a probe to screen a rat genomic library for recombinant phages containing myosin heavy chain sequences. Six clones with approximately 15 k bp inserts each were isolated. Digestion with several restriction enzymes and hybridization of the fractionated DNA with the plasmid probe showed that the clones contained 3 different DNA inserts. Electron microscopy of a heteroduplex made by hybridization of DNA from two clones confirmed that the inserts originated in different genes. Hybridization of size-fractionated ECOR1 digested rat spleen DNA with the cloned probe suggested the existence of at least 5 myosin heavy chain genes. 相似文献
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The sequence of the Saccharomyces cerevisiae gene PHO2 codes for a regulatory protein with unusual aminoacid composition. 总被引:23,自引:4,他引:19 下载免费PDF全文
A new centromere vector for the construction of a Saccharomyces cerevisiae gene library, allowing direct selection for DNA insert, will be described. From that library the gene for the regulatory protein PHO2 involved in PHO5 induction has been cloned by complementation of a pho2 mutation. The complementing activity was shown to be located on a 3.6 kb HindIII fragment. This fragment was used to evict the genomic copy and with appropriate genetic crosses we proved, that the cloned gene is PHO2. The DNA sequence of PHO2 was determined. Analysis of the sequence data uncovered striking homology regions with PHO4, another protein necessary for the induction of PHO5. The relevance of the observed homology will be discussed. 相似文献
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Construction and validation of two metagenomic DNA libraries from Cerrado soil with high clay content 总被引:1,自引:0,他引:1
de Castro AP Quirino BF Allen H Williamson LL Handelsman J Krüger RH 《Biotechnology letters》2011,33(11):2169-2175
A challenge of metagenomic studies is in the extraction and purification of DNA from environmental samples. The soils of the
Cerrado region of Brazil present several technical difficulties to DNA extraction: high clay content (>55% w/w), low pH (4.7)
and high iron levels (146 ppm). Here we describe for the first time the efficient recovery and purification of microbial DNA
associated with these unusual soil characteristics and the construction and validation of two metagenomic libraries: a 150,000
clones library with insert size of approximately 8 kb and a 65,000 clones library with insert size of approximately 35 kb.
The construction of these metagenomic libraries will allow the biotechnological exploitation of the microbial community present
in the soil from this endangered biome. 相似文献
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Instability of bacterial artificial chromosome (BAC) clones containing tandemly repeated DNA sequences. 总被引:6,自引:0,他引:6
The cloning and propagation of large DNA fragments as bacterial artificial chromosomes (BACs) has become a valuable technique in genome research. BAC clones are highly stable in the host, Escherichia coli, a major advantage over yeast artificial chromosomes (YACs) in which recombination-induced instability is a major drawback. Here we report that BAC clones containing tandemly repeated DNA elements are not stable and can undergo drastic deletions during routine library maintenance and DNA preparation. Instability was observed in three BAC clones from sorghum, rice, and potato, each containing distinct tandem repeats. As many as 46% and 74% of the single colonies derived from a rice BAC clone containing 5S ribosomal RNA genes had insert deletions after 24 and 120 h of growth, respectively. We also demonstrated that BAC insert rearrangement can occur in the early stage of library construction and duplication. Thus, a minimum growth approach may not avoid the instability problem of such clones. The impact of BAC instability on genome research is discussed. 相似文献
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G. R. VERHEYEN B. KEMPENAERS T. BURKE M. VAN DEN BROECK C. VAN BROECKHOVEN A. DHONDT 《Molecular ecology》1994,3(2):137-143
We report the isolation of a set of hypervariable minisatellite DNA sequences from a blue tit Parus caeruleus genomic DNA library. In our strategy, we cloned a minisatellite-rich DNA fraction into a charomid vector. The resulting cosmid library was screened with the two minisatellite DNA probes 33.6 and 33.15 for recombinants containing a minisatellite DNA insert. A total of 233 positive clones were isolated. Of 37 clones that have been analysed, nine gave polymorphic signals and can be used as single locus probes (SLPs). Four of the SLPs were investigated in more detail. The number of alleles, the heterozygosity and the mutation rate were estimated. Linkage analysis revealed that two of these loci were linked. The SLPs are of value to studies of the mating system and reproductive success in the blue tit, and may also be useful in population genetic studies. 相似文献
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Haiying Liang Eric G. Fang Jeffrey P. Tomkins Meizhong Luo David Kudrna Hye Ran Kim K. Arumuganathan Shaying Zhao James Leebens-Mack Scott E. Schlarbaum Jo Ann Banks Claude W. dePamphilis Dina F. Mandoli Rod A. Wing John E. Carlson 《Tree Genetics & Genomes》2007,3(3):215-225
Liriodendron tulipifera L., a member of the Magnoliaceae, occupies an important phylogenetic position as a basal angiosperm that has retained numerous
putatively ancestral morphological characters, and thus has often been used in studies of the evolution of flowering plants
and of specific gene families. However, genomic resources for these early branching angiosperm lineages are very limited.
In this study, we describe the construction of a large-insert bacterial artificial chromosome (BAC) library from L. tulipifera. Flow cytometry estimates that this nuclear genome is approximately 1,802 Mbp per haploid genome (±16 SD). The BAC library
contains 73,728 clones, a 4.8-fold genome coverage, with an average insert size of 117 kb, a chloroplast DNA content of 0.2%,
and little to no bacterial sequences nor empty vector content clones. As a test of the utility of this BAC library, we screened
the library with six single/low-copy genic probes. We obtained at least two positive clones for each gene and confirmed the
clones by DNA sequencing. A total of 182 paired end sequences were obtained from 96 of the BAC clones. Using BLAST searches,
we found that 25% of the BAC end sequences were similar to DNA sequences in GenBank. Of these, 68% shared sequence with transposable
elements and 25% with genes from other taxa. This result closely reflected the content of random sequences obtained from a
small insert genomic library for L. tulipifera, indicating that the BAC library construction process was not biased. The first genomic DNA sequences for Liriodendron genes are also reported. All the Liriodendron genomic sequences described in this paper have been deposited in the GenBank data library. The end sequences from shotgun
genomic clones and BAC clones are under accession DU169330–DU169684. Partial sequences of Gigantea, Frigida, LEAFY, cinnamyl alcohol dehydrogenase, 4-coumarate:CoA ligase, and phenylalanine ammonia-lyase genes are under accession DQ223429–DQ223434.
Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. 相似文献
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Yongchang Ouyang Shikun Dai Lianwu Xie M. S. Ravi Kumar Wei Sun Huimin Sun Danling Tang Xiang Li 《Marine biotechnology (New York, N.Y.)》2010,12(3):318-325
Metagenomics is a powerful tool for mining the genetic repositories from environmental microorganisms. Bacteria associated
with marine sponges (phylum Porifera) are rich sources of biologically active natural products. However, to date, few compounds
are discovered from the sponge metagenomic libraries, and the main reason might be the difficulties in recovery of high molecular
weight (HMW) DNA from sponge symbionts to construct large insert libraries. Here, we describe a method to recover HMW bacterial
DNA from diverse sponges with high quality for bacterial artificial chromosome (BAC) library construction. Microorganisms
concentrated from sponges by differential centrifugation were embedded in agarose plugs to lyse out the HMW DNA for recovery.
DNA fragments over 436 kb size were recovered from three different types of sponges, Halichondria sp., Haliclona sp., and Xestospongia sp. To evaluate the recovered DNA quality, the diversity of bacterial DNA comprised in the HMW DNA derived from sponge Halichondria sp. was analyzed, and this HMW DNA sample was also cloned into a shuttle BAC vector between Escherichia coli and Streptomyces sp. The results showed that more than five types of bacterial DNA, i.e., Proteobacteria, Nitrospirae, Cyanobacteria, Planctomycetes,
and unidentified bacteria, had been recovered by this method, and an average 100 kb size insert DNA in a constructed BAC library
demonstrated that the recovered HMW DNA is suitable for metagenomic library construction. 相似文献