棉花BAC文库快速筛选法

目的:构建棉花细菌人工染色体(Bacterial Artificial Chromosome,BAC)文库的快速筛选法,以期从BAC文库中大量、快速、高效筛选出特定BAC克隆,为从事基因组测序、分离和分析特定基因、构建物理图谱及基因图位克隆等生物学技术研究奠定基础。方法:构建了整板、行、列的三维混合池,以菌液PCR为基础,从BAC文库中筛选出含有特定DNA片段的BAC单克隆。结果:从BAC文库的3 456个克隆中,共筛选出16个阳性单克隆,涉及13条染色体、11个SSR标记。结论:该文构建的棉花BAC文库筛选体系,筛选快速、准确,适合从BAC文库中大量筛选BAC单克隆。结合当前的多种BAC文库筛选方法进行探讨,根据不同的实验目的选择更合适的筛选方法和操作步骤。     

基因组细菌人工染色体文库(BAC)的构建及应用

细菌人工染色体 (BAC)是一种承载DNA大片段的克隆载体系统 ,用于人、动物和植物基因组文库构建。BAC具有插入片断大、嵌合率低、遗传稳定性好、易于操作等优点。BAC文库的构建是基因组较大的真核生物基因组学研究的重要基础 ,可用于真核生物重要基因及全基因组物理作图、重要性状基因的图位克隆、基因结构及功能分析。本文主要综述了细菌人工染色体的构建与其鉴定 ,及其在物理图谱构建、图位克隆、转基因技术等研究上的应用。     

稻曲病菌UV-2菌株细菌人工染色体文库构建及分析

【目的】稻曲病(Rice false smut)是由稻曲病菌[Villosiclava virens (Cooke) Tak.]引起的严重危害水稻的真菌病害。构建稻曲病菌UV-2的大片段DNA细菌人工染色体(Bacterial artificial chromosome, BAC)文库, 为致病相关基因的鉴定及在图位克隆、比较基因组学等方面的研究奠定基础。【方法】以幼嫩菌丝为材料制备大分子基因组DNA包埋块, 用Hind III部分酶解后经脉冲凝胶电泳筛选, 回收大片段DNA并与pIndigoBAC536-S 载体连接, 连接产物转化大肠杆菌菌株DH10B T1 Phage-Resistant 细胞后进行蓝白斑筛选, 白色菌落捡入384孔板置于?80 °C低温保存。【结果】成功构建UV-2菌株的高质量、高覆盖度的BAC文库, 该文库共含10 368个克隆, 平均插入片段为124.4 kb, 空载率小于1%, 约覆盖该菌基因组的36.8倍。【结论】克服了真菌大分子基因组DNA制备难控制的技术难题, 建立了首个稻曲病菌的BAC文库。该文库已作为一种公共基因组资源向研究者开放(http://GResource.hzau.edu.cn)。     

温敏核不育水稻5460S细菌人工染色体文库的构建和鉴定

为了构建温敏核不育 (TGMS)基因区域的精细物理图谱并最终分离TGMS基因 ,以温敏核不育水稻 5 46 0S为材料 ,摸索优化了构建植物细菌人工染色体 (BAC)文库的方法 ,构建了一个高质量的BAC文库 .该文库由 1 95 84个克隆构成 ,插入片段平均长度为 1 1 0kb ,相当于水稻单倍体基因组大小的 5倍 ;以分子量分别为 1 40和2 5 0kb的 2个大BAC克隆进行稳定性传代实验 ,经 1 0 0代传代后其插入的DNA片段仍然稳定存在 ;以线粒体和叶绿体基因为探针筛选BAC文库 ,未检验出叶绿体和线粒体DNA的污染 ;以和tms1基因连锁的 3个分子标记作为探针对BAC文库进行了筛选 ,每个探针至少可获得一个阳性克隆 ,利用热不对称性交错PCR(Tail PCR)法成功分离了阳性克隆的左右末端序列 .     

细菌人工染色体文库的构建方法分析

细菌人工染色体是一种承载大片段DNA的新型载体系统,它具有插入片断大、嵌合率低、遗传稳定性好、易于操作等优点.在高等生物基因组文库的构建和基因功能的分析等方面有广泛应用.BAC文库的构建是基因组较大的真核生物基因组学研究的重要基础.介绍了近年来细菌人工染色体文库构建方法上的研究进展,对其中载体的制备和高分子量DNA的制备这两个关键环节作了较深入的探讨.     

基因组文库的研究进展

基因组文库是分子克隆和基因组学的技术基础,它主要经历了噬菌体系列文库、人工染色体系列文库和多元载体系列文库三个阶段。介绍了λ噬菌体文库、cosmid文库、P1噬菌体及PAC文库、fosmid文库、YAC文库、BAC文库、MAC文库、HAC文库、BIBAC文库和TAC文库,罗列了部分文库的研究成果和发展情况,总结了基因组文库的发展进程,并对基因组文库向多基因发展作了展望。     

细菌人工染色体文库的构建及应用

细菌人工染色体（BAC）是第二代大片段DNA的克隆载体系统,具有容量大、嵌合率低、遗传特性稳定、转化效率高、插入片段易回收、操作简便等优点,因而被广泛应用于基因组较大的真核生物基因组研究中,并发挥着前所未有的重要作用。本文综述了BAC的发展,利用此载体构建基因组文库的程序和鉴定方法,及其在物理图谱构建、图位克隆、基因组测序、转基因技术等研究中的应用。     

大片段DNA插入文库的研究进展

基因组DNA大片段插入文库作为基因组学和基因克隆的技术平台,它的发展主要经历了Cosmid文加,YACs文库,BACs文库三个阶段,文中对几种文库作了简单的比较和评价,对大片段文库的应用人了比较详实的介绍。作者根据自己的建库经验,重点介绍了BAC文库的构建。     

细菌人工染色体基因组文库构建关键技术研究

基因组文库是进行分子克隆和基因结构与功能研究的基础,完整覆盖的基因组文库的构建,使基因组任何DNA片段的筛选和获得成为可能.细菌人工染色体(Bacterial Artificial Chromosome,BAC)与其它载体系统相比具有转化效率高、嵌合体少、插入片段易回收,高覆盖率、稳定性强,并且具有易分离和操作等特性等优点而被广泛应用.作为物种保护策略的重要部分,构建我国濒危动植物品种基因组BAC文库,对于其遗传资源的保护和研究具有非常重要的作用.在查阅大量国内外相关资料的基础上,就BAC基因组文库构建过程中栽体的制备、高分子量DNA的制备、大片段DNA的回收、连接与电击转化、基因组BAC文库质量鉴定等几个重点、难点问题进行了详细的论述和分析,对于构建高分子量插入片段、高覆盖率和稳定性的基因组文库提供理论基础与技术支撑.     

细菌人工染色体的研究和应用

细菌人工染色体 (Bacterialartificialchromosome ,BAC)是第二代大片段DNA的克隆载体系统。因其嵌合率低 ,遗传稳定性好 ,重组DNA容易分离和制备 ,转化效率高等 ,弥补了YAC的不足 ,很快在基因组研究中处于中心地位。近年来 ,已有多种BAC载体被构建出来 ,这些BAC载体在复杂基因组大片段文库的构建 ,基因的图位克隆 ,基因组物理图谱的构建 ,基因和基因组测序 ,基因组织结构分析 ,染色体组织和进化 ,以及基因的遗传转化和调控研究中得到了广泛的应用。     

Construction and characterization of a bacterial artificial chromosome library of Sorghum bicolor.

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.     

Construction and characterization of a bacterial artificial chromosome library of thermo-sensitive genie male-sterile rice 5460S

In order to develop a detailed physical map of the thermo-sensitive genie male-sterile (TGMS) gene-encompassing region and finally clone the TGMS gene, a high-quality rice bacterial artificial chromosome (BAC) library from TGMS rice 5460S was constructed. The method of constructing BAC library was examined and optimized. The 5460S library consists of 19 584 BAC clones with an average insert size of 110 kb, which represents about 5 times rice haploid genome equivalents. Rice inserts of up to 140 kb and 250 kb were isolated and appeared stable after 100 generations of serial growth. Hybridization of BAC clones with mitochondrial and chloroplastic genes as probes demonstrated that this library has no organellar contamination. The 5460S library was screened with 3 molecular markers linked to tmsl gene as probes and at least 1 BAC clone was identified with each probe. The insert ends of positive clones were successfully isolated using thermal asymmetric interlaced PCR (TAIL-PCR) technique.     

Mining the metagenome of activated biomass of an industrial wastewater treatment plant by a novel method

Metagenomic libraries herald the era of magnifying the microbial world, tapping into the vast metabolic potential of uncultivated microbes, and enhancing the rate of discovery of novel genes and pathways. In this paper, we describe a method that facilitates the extraction of metagenomic DNA from activated sludge of an industrial wastewater treatment plant and its use in mining the metagenome via library construction. The efficiency of this method was demonstrated by the large representation of the bacterial genome in the constructed metagenomic libraries and by the functional clones obtained. The BAC library represented 95.6 times the bacterial genome, while, the pUC library represented 41.7 times the bacterial genome. Twelve clones in the BAC library demonstrated lipolytic activity, while four clones demonstrated dioxygenase activity. Four clones in pUC library tested positive for cellulase activity. This method, using FTA cards, not only can be used for library construction, but can also store the metagenome at room temperature.     

Construction of Japanese BAC library Yamato-2 (JY2): a set of 330K clone resources of damage-minimized DNA taken from a genetically established Japanese individual

A bacterial artificial chromosome (BAC) library referred to as Yamato-2 (JY2), was constructed from a Japanese individual and contained 330,000 clones. Library construction was based on 2 concepts: Japanese pedigree and non-immortalization. Genomic DNA was extracted from white blood cells from umbilical cord blood of a Japanese male individual. Four traits of the sample, (1) amelogenin DNA, (2) short tandem repeat (STR), (3) mitochondrial DNA (mtDNA), and (4) HLA-allele typing, were investigated to verify attribution of the donor. One of the samples with quite good Japanese characteristics was named JY2 and used as a resource for construction of a BAC library. Amelogenin DNA indicated male. STR indicated Mongoloid. MtDNA suggested haplogroup B, which is different from any other diploid whose sequence has been reported. The HLA gene was classified into east-Asian specific haplotype. These results revealed that JY2 was obtained from a Japanese male. We sequenced both ends of 185,012 BAC clones. By using the BLAST search, BAC end sequences (BESs) were mapped on the human reference sequence provided by NCBI. Inserts of individual BAC clones were mapped with both ends properly placed. As a result, 103,647 BAC clones were successfully mapped. The average insert size of BAC calculated from the mapping information was 130?kb. Coverage and redundancy of the reference sequence by successfully mapped BAC clones were 96.4% and 3.9-fold, respectively. This library will be especially suitable as a Japanese standard genome resource. The availability of an accurate library is indispensable for diagnostics or drug-design based on genome information, and JY2 will provide an accurate sequence of the Japanese genome as an important addition to the human genome.     

Construction of a BAC library and identification of Dmrt1 gene of the rice field eel, Monopterus albus

A bacterial artificial chromosome (BAC) library was constructed using nuclear DNA from the rice field eel (Monopterus albus). The BAC library consists of a total of 33,000 clones with an average insert size of 115 kb. Based on the rice field eel haploid genome size of 600 Mb, the BAC library is estimated to contain approximately 6.3 genome equivalents and represents 99.8% of the genome of the rice field eel. This is first BAC library constructed from this species. To estimate the possibility of isolating a specific clone, high-density colony hybridization-based library screening was performed using Dmrt1 cDNA of the rice field eel as a probe. Both library screening and PCR identification results revealed three positive BAC clones which were overlapped, and formed a contig covering the Dmrt1 gene of 195 kb. By sequence comparisons with the Dmrt1 cDNA and sequencing of first four intron-exon junctions, Dmrt1 gene of the rice field eel was predicted to contain four introns and five exons. The sizes of first and second intron are 1.5 and 2.6 kb, respectively, and the sizes of last two introns were predicted to be about 20 kb. The Dmrt1 gene structure was conserved in evolution. These results also indicate that the BAC library is a useful resource for BAC contig construction and molecular isolation of functional genes.     

Construction of a bacterial artificial chromosome library from the inshore hagfish, Eptatretus burgeri: A resource for the analysis of the agnathan genome

The jawless fish occupy an important phylogenetic position for understanding the evolution of body plans, the origin of adaptive immunity and genome evolution in chordates. We describe here the construction of a large-insert bacterial artificial chromosome (BAC) library from the inshore hagfish, Eptatretus burgeri. The BAC library contains 93,978 clones with an average insert size of 100 kb and is estimated to represent threefold genome-equivalent coverage. The library was organized in three-dimensional pools to facilitate screening by PCR. We have screened this library by PCR and isolated several BAC clones; the average number of positive clones was compatible with the estimated genome coverage of the library. This BAC library, constructed for the first time from the jawless fish, should serve as a useful resource for the scientific community.     

A Large-Insert (130 kbp) Bacterial Artificial Chromosome Library of the Rice Blast FungusMagnaporthe grisea:Genome Analysis, Contig Assembly, and Gene Cloning

Magnaporthe grisea(Hebert) Barr causes rice blast, one of the most devastating diseases of rice (Oryza sativa) worldwide. This fungus is an ideal organism for studying a number of aspects of plant–pathogen interactions, including infection-related morphogenesis, avirulence, and pathogen evolution. To facilitateM. griseagenome analysis, physical mapping, and positional cloning, we have constructed a bacterial artificial chromosome (BAC) library from the rice infecting strain 70-15. A new method was developed for separation of partially digested large-molecular-weight DNA fragments that facilitated library construction with large inserts. The library contains 9216 clones, with an average insert size of 130 kbp (>25 genome equivalents) stored in 384-well microtiter plates that can be double spotted robotically on to a single nylon membrane. Several unlinked single-copy DNA probes were used to screen 4608 clones in the library and an average of 13 (minimum of 6) overlapping BAC clones was found in each case. Hybridization of total genomic DNA to the library and analysis of individual clones indicated that ≈26% of the clones contain single-copy DNA. Approximately 35% of BAC clones contained the retrotransposon MAGGY. The library was used to identify BAC clones containing a adenylate cyclase gene (mac1). In addition, a 550-kbp contig composed of 6 BAC clones was constructed that encompassed two adjacent RFLP markers on chromosome 2. These data show that the BAC library is suitable for genome analysis ofM. grisea.Copies of colony hybridization membranes are available upon request.     

Construction and characterization of a soybean bacterial artificial chromosome library and use of multiple complementary libraries for genome physical mapping

Two plant-transformation-competent large-insert binary clone bacterial artificial chromosome (hereafter BIBAC) libraries were previously constructed for soybean cv. Forrest, using BamHI or HindIII. However, they are not well suited for clone-based genomic sequencing due to their larger ratio of vector to insert size (27.6 kbp:125 kbp). Therefore, we developed a larger-insert bacterial artificial chromosome (BAC) library for the genotype in a smaller vector (pECBAC1), using EcoRI. The BAC library contains 38,400 clones; about 99.1% of the clones have inserts; the average insert size is 157 kbp; and the ratio of vector to insert size is much smaller (7.5 kbp:157 kbp). Colony hybridization with probes derived from several chloroplast and mitochondrial genes showed that 0.89% and 0.45% of the clones were derived from the chloroplast and mitochondrial genomes, respectively. Considering these data, the library represents 5.4 haploid genomes of soybean. The library was hybridized with six RFLP marker probes, 5S rDNA and 18S-5.8S-25S rDNA, respectively. Each RFLP marker hybridized to about six clones, and the 5S and 18S-5.8S-25S rDNA probes collectively hybridized to 402 BACs—about 1.05% of the clones in the library. The BAC library complements the existing soybean Forrest BIBAC libraries by using different restriction enzymes and vector systems. Together, the BAC and BIBAC libraries encompass 13.2 haploid genomes, providing the most comprehensive clone resource for a single soybean genotype for public genome research. We show that the BAC library has enhanced the development of the soybean whole-genome physical map and use of three complementary BAC libraries improves genome physical mapping by fingerprint analysis of most of the clones of the library. The rDNA-containing clones were also fingerprinted to evaluate the feasibility of constructing contig maps of the rDNA regions. It was found that physical maps for the rDNA regions could not be readily constructed by fingerprint analysis, using one or two restriction enzymes. Additional data to fingerprints and/or different fingerprinting methods are needed to build contig maps for such highly tandem repetitive regions and thus, the physical map of the entire soybean genome.     

Construction of a BAC Library for a Defoliating Insect-Resistant Soybean and Identification of Candidate Clones Using a Novel Approach

Positional cloning of an insect-resistance quantitative trait locus (QTL) requires the construction of a large-insert genomic DNA library from insect-resistant genotypes. To facilitate cloning of a major defoliating insect-resistance QTL on linkage group M of the soybean genetic map, a bacterial artificial chromosome (BAC) library for PI 229358 was constructed and characterized. The HindIII BAC library contains 55,296 clones with an average insert size 131 kb. This library represents a 6-fold soybean haploid genome equivalents, allowing a 99.8% probability of recovering any specific sequence of interest in soybean. BAC filters were screened with a genomic DNA probe Sat_258sc2 obtained through genome walking from flanking sequences of a simple sequence repeat (SSR) marker, Sat_258, which links to the insect-resistance QTL. Thirteen BAC clones were identified positive for Sat_258sc2, and two of them were confirmed to carry Sat_258. The results suggest that this library is useful in positional cloning of the major insect-resistance QTL, and the approach presented here can be used to screen a BAC library for a SSR marker without requiring the creation of BAC pools.