用克隆池PCR法筛选小麦-簇毛麦易位系6VS/6AL基因组TAC文库获得抗病基因候选克隆

用根据抗病基因保守区设计的一对简并性引物,从小麦－簇毛麦易位系6VS／6AL cDNA中PCR扩增获得一个具有抗病基因核苷酸结合位点（Nucleotide binding site,NBS）结构特点的DNA片段克隆N7。从小麦－簇毛麦易位系6VS／6AL基因组TAC（Transformation-competent artificial chromosome,TAC）文库的22块96孔板提取所有2112个克隆池（每个池含约1000个克隆）的质粒,再根据N7的核苷酸序列设计一对特异引物,用克隆池PCR（pooled PCR)法经分级筛选从文库中获得一个阳性克隆。以N7为探针,通过Southern杂交证实了该TAC克隆为真正含有抗病候选基因的克隆。研究结果表明克隆池PCR法对克隆数目巨大的基因组文库的筛选很有效。     

Terminal-restriction fragment length polymorphism (T-RFLP) screening of a marine archaeal clone library to determine the different phylotypes

T-RFLP clone characterization (screening) was optimized for a fast and basepair-accurate characterization of clones from marine Archaea collected from the Eastern Mediterranean Sea. Because of the high sensitivity of T-RFLP fingerprinting, a protocol was developed where 10 initial PCR cycles gave detectable terminal fragments from clones. Additionally, forward and reverse primers for PCR were individually labeled and detected simultaneously to assess the suitability of the forward and reverse fragments for T-RFLP screening. Based on independent restriction digests with the tetrameric restriction enzymes HhaI, RsaI and HaeIII to characterize the 49 archaeal clones in our library, the clones were grouped into 13 T-RFLP operational taxonomic units (OTUs). Reverse fragments generally gave less heterogeneous fragments in size. The accuracy of T-RFLP screening was evaluated by sequencing representative clones. Closely related clones ( approximately 97% similarity) could only be resolved with multiple restriction digests where forward and reverse fragments were included in the analysis. All fragments from the clone library were detected in the T-RFLP fingerprint from the complex archaeal community. We found representatives of marine group I, II and III Archaea. Thus, the recently discovered low abundant marine group III Archaea could be clearly differentiated from the other clones in our library and comprised a considerable fraction of the clone library ( approximately 12%). Therefore, our T-RFLP screening approach proved successful in characterizing novel archaeal sequences from the marine environment.     

簇毛麦端体6VS的显微切割及其专化DNA序列的克隆和分析

从簇毛麦(Haynaldia villosa (L.) Schur.)组合CA9211/RW15(6D/6V异代换系)幼胚培养SC2后代中,用原位杂交方法鉴定出T240-6为6VS端体异代换系. 以此为材料,采用微细玻璃针切割法及"单管反应"技术体系,对6VS进行切割分离及LA (Linker adaptor)-PCR扩增.扩增带在100～3 000 bp 之间,大部分集中在600～1 500 bp.利用32P标记的簇毛麦基因组为探针进行Southern杂交,证实扩增产物来源于簇毛麦.扩增产物纯化后,连接到pGEM-T载体上,构建了6VS DNA质粒文库.对文库的分析表明,文库大约有17 000个白色克隆;插入片段分布在100～1 500 bp,平均600 bp.点杂交结果表明,37%克隆有中度到强烈的杂交信号,证明含有中度或高度重复序列;63%克隆有较弱的信号或没有信号,证明为单/低拷贝序列克隆.从文库中获得8个簇毛麦特异克隆,对其中两个克隆pHVMK22和 pHVMK134进行了RFLP分析和序列分析,并利用该探针对小麦抗白粉病基因Pm21进行了检测.RFLP 结果表明,两个克隆一个为低拷贝序列克隆(pHVMK22),另一个为高度重复序列克隆,均为簇毛麦专化DNA序列.以pHVMK22为探针对抗、感病小麦(Triticum aestivum L.)品系的Southern杂交发现抗病品系有一条2 kb的特征带, 该探针可能作为检测抗病基因Pm21的探针.     

Sequence verification as quality-control step for production of cDNA microarrays

To generate cDNA arrays in our core laboratory, we amplified about 2300 PCR products from a human, sequence-verified cDNA clone library. As a quality-control step, we sequenced the PCR products immediately before printing. The sequence information was used to search the GenBank database to confirm the identities. Although these clones were previously sequence verified by the company, we found that only 79% of the clones matched the original database after handling. Our experience strongly indicates the necessity to sequence verify the clones at the final stage before printing on microarray slides and to modify the gene list accordingly.     

Cloning and expression of flavonol synthase from Petunia hybrida

Flavonols are important co-pigments in flower colour and are also essential for pollen tube growth. In petunia, flavonol synthesis is controlled by the Fl locus. Flavonol synthase (FLS) belongs to the 2-oxoglutarate-dependent dioxygenase family. Dioxygenase gene fragments were amplified by PCR on cDNA made from FlFl and flfl flowers using degenerate primers designed from conserved dioxygenase sequences. A petunia petal cDNA library was screened for clones that hybridized more strongly to the Fl PCR products than the fl PCR products. A full-length cDNA clone identified by this screening exhibited FLS activity when expressed in yeast. FLS gene expression is developmentally regulated during flower development. Antisense expression of an FLS cDNA clone in petunia markedly reduced flavonol synthesis in petals. RFLP mapping showed that the FLS gene is linked to Fl , suggesting that Fl is the structural gene for FLS.     

Construction and characterization of a gridded cattle BAC library

A bovine genomic large-insert bacterial artificial chromosome (BAC) library has been constructed from leukocytes of a Holstein-Friesian male. Size fractionated DpnII-digested genomic DNA was ligated to the dephosphorylated BamH1 ends of a pBACe3.6 vector. Approximately 8.3 x 10(4) individual BAC clones were picked into 384-well plates. Two-hundred and sixty-seven randomly chosen clones were characterized by pulsed-field gel electrophoresis (PFGE). The average insert size was 104 kb with a frequency of clones without inserts of 5.5%. Thirty-four BAC clones were mapped by fluorescence in situ hybridization (FISH) to cattle chromosomes. Three showed signals at more than one location, one of them on the centromeric regions of all autosomes, indicating that the clone contains centromeric repeats. A subset of these BAC clones was used for the development of sequence tagged sites. Both subcloning and direct sequencing of the BACs were used for generating sequence tagged site information. The clones from the library were gridded onto high-density membranes, and PCR superpools were produced from the same set of clones. Membranes and superpools are available through the Resource Centre of the German Human Genome Project in Berlin (http:// www.rzpd.de).     

Polymerase chain reaction amplification and sequence analysis of human mutant adenine phosphoribosyltransferase genes: the nature and frequency of errors caused by Taq DNA polymerase

Using the polymerase chain reaction (PCR) with Taq DNA polymerase, we have amplified a 2.4-kb fragment of genomic DNA containing the adenine phosphoribosyltransferase (APRT) gene from patients with APRT deficiency. Several clones from each patient were sequenced after subcloning the PCR product into M13mp18. Selected regions of the amplified fragment were also sequenced directly. This enabled us to distinguish PCR-induced errors from endogenous mutations and polymorphisms in each clone. 44 PCR errors were found in a total of 57,94 kb of DNA sequenced from 25 clones from 7 patients. All the errors were due to the PCR process and not to subcloning, as shown by sequence analysis of 5 APRT-positive clones isolated from a phage genomic library.     

Diversity of thermophilic and non-thermophilic Crenarchaeota at 80 degrees C

A hot spring in the solfataric field of Pisciarelli (Naples-Italy) was analysed for Archaeal diversity. Total DNA was extracted from the environment, archaeal 16S rRNA genes were amplified with Archaea specific primers, and a clone library consisting of 201 clones was established. The clones were grouped in 10 different groups each representing a specific band pattern using restriction fragment length polymorphism (RFLP). Members of all 10 groups were sequenced and phylogenetically analyzed. Surprisingly, a high abundance of clones belonging to non-thermophilic Crenarchaeal clusters were detected together with the thermophilic archaeon Acidianus infernus in this thermophilic environment. Neither Sulfolobus species nor other hyperthermophilic Crenarchaeota were detected in the clone library. The relative abundance of the sequenced clones was confirmed by terminal restriction fragment analyses. Amplification of 16S rRNA genes from Archaea transferred from the surrounding environment was considered negligible because DNA from non-thermophilic Crenarchaeota incubated under conditions similar to the solfatara could not be PCR amplified after 5 min.     

Diversity of nitrite reductase genes in "Candidatus Accumulibacter phosphatis"-dominated cultures enriched by flow-cytometric sorting

"Candidatus Accumulibacter phosphatis" is considered a polyphosphate-accumulating organism (PAO) though it has not been isolated yet. To reveal the denitrification ability of this organism, we first concentrated this organism by flow cytometric sorting following fluorescence in situ hybridization (FISH) using specific probes for this organism. The purity of the target cells was about 97% of total cell count in the sorted sample. The PCR amplification of the nitrite reductase genes (nirK and nirS) from unsorted and sorted cells was performed. Although nirK and nirS were amplified from unsorted cells, only nirS was detected from sorted cells, indicating that "Ca. Accumulibacter phosphatis" has nirS. Furthermore, nirS fragments were cloned from unsorted (Ba clone library) and sorted (Bd clone library) cells and classified by restriction fragment length polymorphism analysis. The most dominant clone in clone library Ba, which represented 62% of the total number of clones, was not found in clone library Bd. In contrast, the most dominant clone in clone library Bd, which represented 59% of the total number of clones, represented only 2% of the total number of clones in clone library Ba, indicating that this clone could be that of "Ca. Accumulibacter phosphatis." The sequence of this nirS clone exhibited less than 90% similarity to the sequences of known denitrifying bacteria in the database. The recovery of the nirS genes makes it likely that "Ca. Accumulibacter phosphatis" behaves as a denitrifying PAO capable of utilizing nitrite instead of oxygen as an electron acceptor for phosphorus uptake.     

口虾蛄肠道细菌种群多样性的分子分析

目的探讨口虾蛄肠道细菌种群的多样性。方法通过不依赖于分离培养的分子生物学分析方法,以直接提取虾蛄肠道细菌的总DNA为模板经过PCR扩增16S DNA,然后经与T载体连接建立质粒文库。用限制性内切酶(BsuRⅠ和Hin6Ⅰ)对阳性克隆的PCR扩增产物进行限制性酶切片段长度多态性(RFLP)分析,选取有代表性的克隆进行测序。结果16S DNA序列通过CLUSTALX进行多序列比对及NCBI数据库中的BLAST分析后发现口虾蛄肠道细菌主要分成4类:未培养细菌,未培养支原体科细菌,未培养δ变形菌和马特斯支原体。结论口虾蛄肠道细菌种类较为简单,且多为未培养的细菌。     

Analysis of microbial communities using culture-dependent and culture-independent approaches in an anaerobic/aerobic SBR reactor

Comparative analysis of microbial communities in a sequencing batch reactor which performed enhanced biological phosphorus removal (EBPR) was carried out using a cultivation-based technique and 16S rRNA gene clone libraries. A standard PCR protocol and a modified PCR protocol with low PCR cycle was applied to the two clone libraries of the 16S rRNA gene sequences obtained from EBPR sludge, respectively, and the resulting 424 clones were analyzed using restriction fragment length polymorphisms (RFLPs) on 16S rRNA gene inserts. Comparison of two clone libraries showed that the modified PCR protocol decreased the incidence of distinct fragment patterns from about 63% (137 of 217) in the standard PCR method to about 34% (70 of 207) under the modified protocol, suggesting that just a low level of PCR cycling (5 cycles after 15 cycles) can significantly reduce the formation of chimeric DNA in the final PCR products. Phylogenetic analysis of 81 groups with distinct RFLP patterns that were obtained using the modified PCR method revealed that the clones were affiliated with at least 11 phyla or classes of the domain Bacteria. However, the analyses of 327 colonies, which were grouped into just 41 distinct types by RFLP analysis, showed that they could be classified into five major bacterial lineages: alpha, beta, gamma- Proteobacteria, Actinobacteria, and the phylum Bacteroidetes, which indicated that the microbial community yielded from the cultivation-based method was still much simpler than that yielded from the PCR-based molecular method. In this study, the discrepancy observed between the communities obtained from PCR-based and cultivation-based methods seems to result from low culturabilities of bacteria or PCR bias even though modified culture and PCR methods were used. Therefore, continuous development of PCR protocol and cultivation techniques is needed to reduce this discrepancy.     

犬2型腺病毒E1转化细胞的建立

为建立能表达犬2型腺病毒(CAV2)E1基因的辅助细胞,本研究用含CAV2E1基因的重组质粒pcE1t对DK细胞进行转染,经G418的筛选,得到26个细胞克隆。用CAV2E1A特异引物对各细胞克隆进行PCR和RTPCR检测,PCR结果表明转染后的细胞克隆都能扩增出536bp的特异性片段,但仅有6株为RTPCR强阳性,能扩增出652bp的特异带。再对RTPCR强阳性的细胞克隆进行Westernblot分析,最终挑选到一个既能转录E1A基因,又能表达E1B19kD蛋白的克隆细胞株(DKE1)。     

An Alternative Approach in Gateway® Cloning when the Bacterial Antibiotic Selection Cassettes of the Entry Clone and Destination Vector are the Same

The Gateway^® recombination technology has revolutionized the method of gene cloning for functional analyses and high-throughput ORFeome projects. In general, Gateway cloning is highly efficient because after LR recombination and bacterial transformation, only cells containing the recombinant destination clone are selected on an antibiotic selection plate. However, when the antibiotic resistance gene for bacterial selection is the same in the entry and destination vectors, the direct selection of recombinant destination clones on an antibiotic plate is difficult. Here, we demonstrate an efficient and comprehensive approach to obtain positive destination clones directly on an antibiotic selection plate in this situation. The strategy involves polymerase chain reaction (PCR)-mediated amplification of the entry clone using entry vector-specific primers that bind outside the attL sequences and the subsequent use of this purified PCR product for LR recombination with the destination vector. Our results suggest that cloning of linear DNA fragments into circular destination vectors through LR recombination is an efficient method for inserts up to 7 kb in size. Using this approach, the yield of colony PCR positive destination clones was 100 % for genes of various sizes tested in our experiments.     

A molecular approach to search for diversity among bacteria in the environment

Molecular 16S rDNA-based techniques were applied to a peat sample from northern Germany in order to investigate the bacterial diversity present and compare the clone sequences with those obtained from similar studies on other terrestrial samples. Genomic DNA was extracted from the peat matrix by a direct lysis procedure. 16S rRNA genes were amplified using PCR primers targeting conserved regions of bacterial 16S rDNA. 16S rDNA fragments were blunt end cloned into a plasmid vector and the resulting clone library of 262 sequences was screened by hybridization with different oligonucleotide probes and sequence analysis of randomly selected clones. The 16S rDNA insert of 76 clones was partially sequenced. Clones identified either by hybridization or by sequence analysis fell into three phyla. As judged by hybridization with a specific oligonucleotide probe, 42% of the clones represented members of the alpha subclass of Proteobacteria. Twenty-five of these clones were selected randomly for sequence analysis; none could be assigned to any of the known genera of this subclass. The second largest clone group comprises 15% of the clones and clusters aroundAcidimicrobium ferrooxidans andRubrobacter radiotolerans, both of which are remotely related to members of the order Actinomycetales. The third major clone cluster (10%) was moderately to remotely related to theAcidobacterium capsulatum phylum. Of the additional clones sequenced, a few could be assigned to other subclasses ofProteobacteria, theVerrucomicrobium phylum and the phylum of spirochetes. Comparison of the results presented here with those from other environments reveals a significant number of common clone clusters. As the vast majority of sequences retrieved from any of the marine and terrestrial samples investigated so far by molecular methods indicate the presence of novel bacterial species it can be assumed that a huge, as yet untapped biotechnological potential is present in the environment.     

Two-dimensional screening of the Wageningen chicken BAC library

We have constructed a Bacterial Artificial Chromosome (BAC) library that provides 5.5-fold redundant coverage of the chicken genome. The library was made by cloning partial HindIII-digested high-molecular-weight (HMW) DNA of a female White Leghorn chicken into the HindIII site of the vector pECBAC1. Several modifications of standard protocols were necessary to clone efficiently large partial HindIII DNA fragments. The library consists of 49,920 clones arranged in 130 384-well plates. An average insert size of 134 kb was estimated from the analysis of 152 randomly selected BAC clones. The average number of NotI restriction sites per clone was 0.77. After individual growth, DNA was isolated of the pooled clones of each 384-well plate, and subsequently DNA of each plate was isolated from the individual row and column pools. Screening of the Wageningen chicken BAC library was performed by two-dimensional PCR with 125 microsatellite markers. For 124 markers at least one BAC clone was obtained. FISH experiments of 108 BAC clones revealed chimerism in less than 1%. The number of different BAC clones per marker present in the BAC library was examined for 35 markers which resulted in a total of 167 different BAC clones. Per marker the number of BAC clones varied from 1 to 11, with an average of 4.77. The chicken BAC library constitutes an invaluable tool for positional cloning and for comparative mapping studies. Received: 26 October 1999 / Accepted: 6 January 2000     

Metagenome microarray for screening of fosmid clones containing specific genes

A critical step in the process of metagenome analysis is to screen for clones that contain specific genes among a large number of clones. To form one of the sequence-based screening tools of a metagenome library, we designed a format of microarray [metagenome microarray (MGA)] that is arrayed with fosmid library clone DNA samples on a glass slide. We evaluated the MGA using random prime labeled fluorescent probes prepared from PCR products of the target gene and found that we could obtain specific hybridization signals only for the fosmid clone that contained the target gene. We found that the detection limit of the MGA was c. 10 ng microL(-1) of fosmid clone DNA, and that the MGA-based hybridization was quantitative within a concentration range of 10-200 ng microL(-1) of fosmid clone DNA. We used the MGA successfully to identify two fosmid clones that contained 16S rRNA genes from a fosmid library from the sediment of the East Sea, Korea. In conclusion, we have demonstrated that the MGA can be used for screening for fosmid clones containing specific genes in a metagenome library, and that this technology has potential application as a high-throughput metagenome screening tool.     

二代测序应用于检测GT1-7细胞中KISS1和GnRH启动子的DNA甲基化状态的研究

目的:利用二代测序技术检测GT1-7细胞中KISS1和GnRH基因启动子范围内的甲基化状态,并用金标准的亚硫酸氢盐修饰后的克隆测序作为对照,比较二代测序与金标准克隆测序在研究DNA甲基化检测中的差别。方法:提取GT1-7细胞基因组DNA并进行亚硫酸氢盐处理。进行巢式PCR,将PCR产物进行二代测序。同时采用金标准的亚硫酸氢盐修饰后克隆测序的方法作为对照,对相同批次的PCR产物进行克隆测序。结果:PCR产物二代测序结果表明KISS1和GnRH两个基因的27个CpG甲基化位点信息完整,结果准确。挑取10个克隆进行一代测序结果表明序列无丢失,KISS1和GnRH两个基因的27个CpG甲基化位点信息完整。结论:利用高通量的二代测序技术能够有效的对DNA甲基化的PCR产物进行检测,二代测序和克隆测序都是研究DNA甲基化的有效方法,但前者与克隆测序相比每一个读取序列(reads)都相当于一个单克隆,且二代测序每个区段得到成百上千个reads,因此二代测序结果更加精确。     

酵母双杂交相关方法的改良及应用

对酵母双杂交实验过程中较为耗时的阳性克隆鉴定过程进行改进,以期建立一种快速有效的鉴定方法。分别采用液氮冻融法、超声破碎法、渗透压破壁法以及煮沸裂解法裂解酵母细胞,获得质粒作为PCR模板,直接测序鉴定筛选到的相互作用蛋白。以液氮冻融法和超声破碎法裂解细胞获得的质粒为模板进行PCR,得到特异的产物,测序鉴定结果明确,与经典的鉴定方法相比效果相当,但更加经济快捷;而渗透压破壁法和煮沸裂解法则效果不好。说明前两种方法可代替常规方法用于阳性克隆的鉴定,从而加快酵母双杂交实验中大量阳性克隆的筛查工作。