细菌人工染色体基因组文库构建方法的改进

目的:建立一种改进的更简便、易操作的细菌人工染色体(BAC)文库构建方法。方法:在构建猪霍乱沙门氏菌基因组大片段DNA的BAC文库时,对改进的基因组BAC文库构建方法和常规的BAC文库构建方法进行比较。结果:利用改进的方法可简便快速地构建猪霍乱沙门氏菌基因组BAC文库。结论:使用2种方法构建BAC文库,其转化效率,以及在BAC克隆中插入的DNA片段的大小和BAC克隆的稳定性等都相同,从而表明改进的方法更简单、更方便,它能使BAC文库的构建更为高效。     

猪基因组文库的构建及其生长激素基因的分离

本工作以长白猪为材料,分别用Charon28及EMBL3为载体,构建了猪的基因组文库。用牛生长激素基因为探针对基因文库进行筛选,由两个基因文库中各获得一个阳性克隆λPGH1,λPGH2。其后,以质粒pUCl9为载体对λPGHl进行了亚克隆。通过对亚克隆pPGH的酶切图谱及其Southern杂交结果的分折表明,在pPGH中含有完整的猪生长激素基因。     

苜蓿核基因组文库的构建及其实验方法的几点改进

目前,基因文库的构建方法虽已逐步普及,但仍有不少实验因最终未能从构建的文库中钓出目的基因而告失败。其主要原因是构建的文库没有达到预期的要求——真实有效.我们对核 DNA 分离和载体左右臂制备等的常用方法进行了改进,构建了苜蓿的核基因组文库,其容量测定、重组子鉴定及初步的杂交筛选都证明该文库真实有效。苜蓿(Medicago sativa L.)DNA 的制备参照 Shure 等(1983)和 Ausubel 等     

一种存放大基因组文库的新方法

用野生一粒小麦为材料,以细菌人工染色体（pECBAC1)为载体构建了细菌人工染色体克隆混合池（Bacterial artificial chromosome pool）,每池100个克隆。经初步验证,池中靶克隆经12h的培养后仍稳定存活。克隆之间的竞争实验仍在进行当中。     

一种动物基因组DNA提取方法的改进

介绍一种动物基因组ＤＮＡ提取方法。该方法具有简便、快速、实用的特点,所获得的ＤＮＡ数量和质量都很高,可用于各种分子生物学实验。     

莱茵衣藻基因组文库的构建

以莱菌衣藻CC-849为材料,提取基因组DNA,利用BamHⅠ和BglⅡ对基因组DNA进行酶解,获得了可用于构建基因组文库的6-12 kb的基因组片段,并浓缩至200 ng/μL。该片段与λDNA载体连接,经噬菌体蛋白包装、侵染大肠杆菌XL1-blue后,获得了莱菌衣藻基因组文库。该文库的滴度为2.12×10~5 pfu/mL,共有转化子4.26×10~4个,插入片段的平均长度约为9kb,扩增后基因组文库滴度为9.5×10~6 pfu/mL。     

基因组文库的构建和池化筛选

作为物种保护策略的重要部分,建立濒危畜禽的基因组文库,可有效保存濒危畜禽种质资源。BAC(bacterialartificial chromosome)文库具有高容量、遗传特性稳定和嵌合体少等优点,因而被用于畜禽基因组文库的构建。对BAC文库的构建方法和文库池化筛选系统作一综述。     

不吸水链霉菌梧州新亚种基因组文库的构建

以不吸水链霉菌梧州新亚种为材料,提取的总基因组DNA经Sau3 AI不完全酶切。回收20-30kbDNA酶切片段,与经Hpal和BamHI酶切的粘粒载体pKC505进行连接,将连接产物用噬菌体包装蛋白包装。侵染大肠埃希菌DH5α。在舍有安普霉素的LB平板上培养,所得转化子数为12000个,构建成不吸水链霉菌梧州新亚种的基因组文库。以不吸水链霉菌染色体基因组大小为7Mb计算,概括了不吸水链霉菌梧州新亚种约99％的基因组,达到了建库要求的理论值。未扩增文库的滴度为1．2×10^8pfu／L,扩增文库的滴度为4．8×10^11pfu／L,包装效率为每微克DNA1．2×10^5个转化子。随机挑取菌落,提取重组质粒进行酶切电泳分析,均有外源片段插入。基因文库的构建为进一步深入研究梧宁霉素生物合成基因结构及功能奠定基础。     

Construction of a full bacterial artificial chromosome （BAC） library of Oryza sativa genome

We have constructed a full BAC library for the superior early indica variety of Oryza sativa,Guang Lu Ai 4.The MAX Efficiency DH10B with increased stability of inserts was used as BAC host cells.The potent pBelo BACII with double selection markers was used as cloning vector.The cloning efficiency we have reached was as high as 98%,and the transformation efficiency was raised up to 10^6 transformants/μg of large fragment DNA.The BAC recombinant transformants were picked at random and analyzed for the size of inserts,which turned out to be of 120 kb in length on average.We have obtained more than 20,000 such BAC clones.According to conventional probability equation,they covered the entire rice genome of 420,000 kb in length.The entire length of inserts of the library obtained has the 5-to 6-fold coverage of the genome.To our knowledge,this is the first reported full BAC library for a complex genome.     

Construction and characterisation of a gridded chicken cosmid library with four-fold genomic coverage

Gridded genomic libraries are crucial for the positional candidate gene approach. For this purpose we constructed a gridded genomic library from a female chicken using the vector sCos 1. About 110 000 cosmid clones were grown and replicated in 384-well plates. An average insert size of 39 kb was calculated from the analysis of 68 randomly selected clones. No chimerism could be observed from 31 in situ hybridisations. One replica of the library (number 125) has been transferred to the Resource Centre/Primary Database (RZPD) of the German Human Genome Project (DHGP). The whole library was gridded onto four nylon filters at high density for efficient identification of cosmid clones by colony hybridisation. Twenty-two probes were used for screening the library and each of them gave at least one positive signal. This result is in good agreement with a four-fold coverage of the genome as estimated from the insert length and number of recombinant clones. This library provides a powerful tool for rapid physical mapping and complex analysis of the chicken genome.     

Construction of a bacterial artificial chromosome library containing large Eco RI and Hin dIII genomic fragments of lettuce

 Existing bacterial artificial chromosome (BAC) vectors were modified to have unique EcoRI cloning sites. This provided an additional site for generating representative libraries from genomic DNA digested with a variety of enzymes. A BAC library of lettuce was constructed following the partial digestion of genomic DNA with HindIII or EcoRI. Several experimental parameters were investigated and optimized. The BAC library of over 50,000 clones, representing one to two genome equivalents, was constructed from six ligations; average insert sizes for each ligation varied between 92.5 and 142 kb with a combined average insert size of 111 kb. The library was screened with markers linked to disease resistance genes; this identified 134 BAC clones from four regions containing resistance genes. Hybridization with low-copy genomic sequences linked to resistance genes detected fewer clones than expected from previous estimates of genome size. The lack of hybridization to chloroplast and mitochondrial sequences demonstrated that the library was predominantly composed of nuclear DNA. The unique EcoRI site in the BAC vector should allow the integration of BAC cloning with other technologies that utilize EcoRI digestion, such as AFLP^TM markers and RecA-assisted restriction endonuclease (RARE) cleavage, to clone specific large EcoRI fragments from genomic DNA. Received: 5 August 1996 / Accepted: 23 August 1996     

Construction of chickpea genomic library

For construction of chickpea genomic library, DNA was isolated, purified on CsCl gradient and size fractionated into 15-20 Kb fragments after restriction with Sau 3A. These fragments were ligated to phage lambda (EMBL-3) vector and the recombinant molecules packaged in vitro into viable phage particles. The recombinant phages were obtained as phages on a P2 lysogen of E. coli (Spi- selection) and amplified to establish a permanent library. This is the first report of the construction of chickpea genomic library.     

Homology-independent genome integration enables rapid library construction for enzyme expression and pathway optimization in Yarrowia lipolytica

Yarrowia lipolytica is an important oleaginous industrial microorganism used to produce biofuels and other value-added compounds. Although several genetic engineering tools have been developed for Y. lipolytica, there is no efficient method for genomic integration of large DNA fragments. In addition, methods for constructing multigene expression libraries for biosynthetic pathway optimization are still lacking in Y. lipolytica. In this study, we demonstrate that multiple and large DNA fragments can be randomly and efficiently integrated into the genome of Y. lipolytica in a homology-independent manner. This homology-independent integration generates variation in the chromosomal locations of the inserted fragments and in gene copy numbers, resulting in the expression differences in the integrated genes or pathways. Because of these variations, gene expression libraries can be easily created through one-step integration. As a proof of concept, a LIP2 (producing lipase) expression library and a library of multiple genes in the β-carotene biosynthetic pathway were constructed, and high-production strains were obtained through library screening. Our work demonstrates the potential of homology-independent genome integration for library construction, especially for multivariate modular libraries for metabolic pathways in Y. lipolytica, and will facilitate pathway optimization in metabolic engineering applications.     

Construction of a genomic library using high-molecular-weight DNA prepared from peach leaves

A method for constructing a total genomic library of peach DNA is presented. DNA of 200 kb or more is efficiently isolated from relatively large amounts of peach leaves (15 to at least 50 g) and used to construct the library in bacteriophage λ.