SuperSAGE array: the direct use of 26-base-pair transcript tags in oligonucleotide arrays

We developed a new platform for genome-wide gene expression analysis in any eukaryotic organism, which we called SuperSAGE array. The SuperSAGE array is a microarray onto which 26-bp oligonucleotides corresponding to SuperSAGE tag sequences are directly synthesized. A SuperSAGE array combines the advantages of the highly quantitative SuperSAGE expression analysis with the high-throughput microarray technology. We demonstrated highly reproducible gene expression profiling by the SuperSAGE array for 1,000 genes (tags) in rice. We also applied this technology to the detailed study of expressed genes identified by SuperSAGE in Nicotiana benthamiana, an organism for which sufficient genome sequence information is not available. We propose that the SuperSAGE array system represents a new paradigm for microarray construction, as no genomic or cDNA sequence data are required for its preparation.     

Capturing genomic signatures of DNA sequence variation using a standard anonymous microarray platform

Comparative genomics, using the model organism approach, has provided powerful insights into the structure and evolution of whole genomes. Unfortunately, only a small fraction of Earth's biodiversity will have its genome sequenced in the foreseeable future. Most wild organisms have radically different life histories and evolutionary genomics than current model systems. A novel technique is needed to expand comparative genomics to a wider range of organisms. Here, we describe a novel approach using an anonymous DNA microarray platform that gathers genomic samples of sequence variation from any organism. Oligonucleotide probe sequences placed on a custom 44 K array were 25 bp long and designed using a simple set of criteria to maximize their complexity and dispersion in sequence probability space. Using whole genomic samples from three known genomes (mouse, rat and human) and one unknown (Gonystylus bancanus), we demonstrate and validate its power, reliability, transitivity and sensitivity. Using two separate statistical analyses, a large numbers of genomic ‘indicator’ probes were discovered. The construction of a genomic signature database based upon this technique would allow virtual comparisons and simple queries could generate optimal subsets of markers to be used in large-scale assays, using simple downstream techniques. Biologists from a wide range of fields, studying almost any organism, could efficiently perform genomic comparisons, at potentially any phylogenetic level after performing a small number of standardized DNA microarray hybridizations. Possibilities for refining and expanding the approach are discussed.     

Chromatin structure analysis enables detection of DNA insertions into the mammalian nuclear genome

BackgroundGenetically modified organisms (GMOs) have numerous biomedical, agricultural and environmental applications. Development of accurate methods for the detection of GMOs is a prerequisite for the identification and control of authorized and unauthorized release of these engineered organisms into the environment and into the food chain. Current detection methods are unable to detect uncharacterized GMOs, since either the DNA sequence of the transgene or the amino acid sequence of the protein must be known for DNA-based or immunological-based detection, respectively.MethodsHere we describe the application of an epigenetics-based approach for the detection of mammalian GMOs via analysis of chromatin structural changes occurring in the host nucleus upon the insertion of foreign or endogenous DNA.ResultsImmunological methods combined with DNA next generation sequencing enabled direct interrogation of chromatin structure and identification of insertions of various size foreign (human or viral) DNA sequences, DNA sequences often used as genome modification tools (e.g. viral sequences, transposon elements), or endogenous DNA sequences into the nuclear genome of a model animal organism.ConclusionsThe results provide a proof-of-concept that epigenetic approaches can be used to detect the insertion of endogenous and exogenous sequences into the genome of higher organisms where the method of genetic modification, the sequence of inserted DNA, and the exact genomic insertion site(s) are unknown.General significanceMeasurement of chromatin dynamics as a sensor for detection of genomic manipulation and, more broadly, organism exposure to environmental or other factors affecting the epigenomic landscape are discussed.     

Y enriched and Y specific DNA sequences from the genome of the mediterranean fruit fly,Ceratitis capitata

DNA sequences that are enriched or specific to the genome of the male medfly, Ceratitis capitata, have been isolated using a differential hybridization approach. Twelve phage clones from a genomic library have been identified that consistently display more intense hybridization with a genomic DNA probe from males as opposed to one from females. Southern DNA blot analysis reveals that these recombinant clones contain at least one EcoRI fragment that is either specific to the male genome, or more highly represented in it, as compared with the female genome. These EcoRI fragments, when used as probes, all generate a similar pattern of intense multiple bands in genomic DNA of males. This suggests the presence of repetitive sequences that are at least partially homologous in these regions of the genome that are specific to or enriched in males. In situ hybridization to mitotic chromosomes confirms a Y chromosomal origin for the male specific repetitive sequences. Data on the genomic organization, representation and evolutionary conservation of these sequences that are specific to or enriched in males are presented. Studies of the genomic organization and representation of flanking sequences that are not male specific are presented as well.by R. Appels     

Libraries for genomic SELEX.

An increasing number of proteins are being identified that regulate gene expression by binding specific nucleic acidsin vivo. A method termed genomic SELEX facilitates the rapid identification of networks of protein-nucleic acid interactions by identifying within the genomic sequences of an organism the highest affinity sites for any protein of the organism. As with its progenitor, SELEX of random-sequence nucleic acids, genomic SELEX involves iterative binding, partitioning, and amplification of nucleic acids. The two methods differ in that the variable region of the nucleic acid library for genomic SELEX is derived from the genome of an organism. We have used a quick and simple method to construct Escherichia coli, Saccharomyces cerevisiae, and human genomic DNA PCR libraries that can be transcribed with T7 RNA polymerase. We present evidence that the libraries contain overlapping inserts starting at most of the positions within the genome, making these libraries suitable for genomic SELEX.     

Specific single-cell isolation and genomic amplification of uncultured microorganisms

We in this study describe a new method for genomic studies of individual uncultured prokaryotic organisms, which was used for the isolation and partial genome sequencing of a soil archaeon. The diversity of Archaea in a soil sample was mapped by generating a clone library using group-specific primers in combination with a terminal restriction fragment length polymorphism profile. Intact cells were extracted from the environmental sample, and fluorescent in situ hybridization probing with Cy3-labeled probes designed from the clone library was subsequently used to detect the organisms of interest. Single cells with a bright fluorescent signal were isolated using a micromanipulator and the genome of the single isolated cells served as a template for multiple displacement amplification (MDA) using the Phi29 DNA polymerase. The generated MDA product was afterwards used for 16S rRNA gene sequence analysis and shotgun-cloned for additional genomic analysis. Sequence analysis showed >99% 16S rRNA gene homology to soil crenarchaeotal clone SCA1170 and shotgun fragments had the closest match to a crenarchaeotal BAC clone previously retrieved from a soil sample. The system was validated using Methanothermobacter thermoautotrophicus as single-cell test organism, and the validation setup produced 100% sequence homology to the ten tested regions of the genome of this organism.     

A Method for Construction, Cloning and Expression of Intron-Less Gene from Unannotated Genomic DNA

The present century has witnessed an unprecedented rise in genome sequences owing to various genome-sequencing programs. However, the same has not been replicated with cDNA or expressed sequence tags (ESTs). Hence, prediction of protein coding sequence of genes from this enormous collection of genomic sequences presents a significant challenge. While robust high throughput methods of cloning and expression could be used to meet protein requirements, lack of intron information creates a bottleneck. Computational programs designed for recognizing intron–exon boundaries for a particular organism or group of organisms have their own limitations. Keeping this in view, we describe here a method for construction of intron-less gene from genomic DNA in the absence of cDNA/EST information and organism-specific gene prediction program. The method outlined is a sequential application of bioinformatics to predict correct intron–exon boundaries and splicing by overlap extension PCR for spliced gene synthesis. The gene construct so obtained can then be cloned for protein expression. The method is simple and can be used for any eukaryotic gene expression.     

Mercury‐associated DNA hypomethylation in polar bear brains via the LUminometric Methylation Assay: a sensitive method to study epigenetics in wildlife

In this paper we describe a novel approach that may shed light on the genomic DNA methylation of organisms with non‐resolved genomes. The LUminometric Methylation Assay (LUMA) is permissive for genomic DNA methylation studies of any genome as it relies on the use of methyl‐sensitive and ‐insensitive restriction enzymes followed by polymerase extension via Pyrosequencing technology. Here, LUMA was used to characterize genomic DNA methylation in the lower brain stem region from 47 polar bears subsistence hunted in central East Greenland between 1999 and 2001. In these samples, average genomic DNA methylation was 57.9% ± 6.69 (SD; range was 42.0 to 72.4%). When genomic DNA methylation was related to brain mercury (Hg) exposure levels, an inverse association was seen between these two variables for the entire study population (P for trend = 0.17). After dichotomizing animals by gender and controlling for age, a negative trend was seen amongst male animals (P for trend = 0.07) but no associations were found in female bears. Such sexually dimorphic responses have been found in other toxicological studies. Our results show that genomic DNA methylation can be quantitatively studied in a highly reproducible manner in tissue samples from a wild organism with a non‐resolved genome. As such, LUMA holds great promise as a novel method to explore consequential questions across the ecological sciences that may require an epigenetic understanding.     

Isolation and characterization of a species-specific DNA probe for Candida albicans.

As a model for the isolation of species-specific sequences of DNA, we isolated and characterized a species-specific DNA fragment from the Candida albicans genome. We used a series of differential colony hybridization experiments, in which a C. albicans genomic library was hybridized with genomic DNA probes from related organisms to minimize the number of potentially specific C. albicans DNA fragments to be tested. Six clones were tested by dot blot analysis, and one of them, a 1348 bp EcoRI DNA fragment, was confirmed as specific for C. albicans. This species-specific fragment could be utilized as a DNA probe for rapid, sensitive, and specific diagnosis of C. albicans DNA in clinical specimens.     

Comparative genomics: the key to understanding the Human Genome Project

The sequencing of the human genome is well underway. Technology has advanced, such that the total genomic sequence is possible, along with an extensive catalogue of genes via comprehensive cDNA libraries. With the recent completion of the Saccharomyces cerevisiae sequencing project and the imminent completion of that of Caenorhabditis elegans, the most frequently asked question is how much can sequence data alone tell us? The answer is that that a DNA sequence taken in isolation from a single organism reveals very little. The vast majority of DNA in most organisms is noncoding. Protein coding sequences or genes cannot function as isolated units without interaction with noncoding DNA and neighboring genes. This genomic environment is specific to each organism. In order to understand this we need to look at similar genes in different organisms, to determine how function and position has changed over the course of evolution. By understanding evolutionary processes we can gain a greater insight into what makes a gene and the wider processes of genetics and inheritance. Comparative genomics (with model organisms), once the poor relation of the human genome project, is starting to provide the key to unlock the DNA code.     

Design and testing of 'genome-proxy' microarrays to profile marine microbial communities

Microarrays are useful tools for detecting and quantifying specific functional and phylogenetic genes in natural microbial communities. In order to track uncultivated microbial genotypes and their close relatives in an environmental context, we designed and implemented a 'genome-proxy' microarray that targets microbial genome fragments recovered directly from the environment. Fragments consisted of sequenced clones from large-insert genomic libraries from microbial communities in Monterey Bay, the Hawaii Ocean Time-series station ALOHA, and Antarctic coastal waters. In a prototype array, we designed probe sets to 13 of the sequenced genome fragments and to genomic regions of the cultivated cyanobacterium Prochlorococcus MED4. Each probe set consisted of multiple 70-mers, each targeting an individual open reading frame, and distributed along each approximately 40-160 kbp contiguous genomic region. The targeted organisms or clones, and close relatives, were hybridized to the array both as pure DNA mixtures and as additions of cells to a background of coastal seawater. This prototype array correctly identified the presence or absence of the target organisms and their relatives in laboratory mixes, with negligible cross-hybridization to organisms having 相似文献   

CRISPR/Cas9系统在基因组DNA片段编辑中的应用

源于细菌和古菌的Ⅱ型成簇规律间隔短回文重复系统[Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9),CRISPR/Cas9]近年被改造成为基因组定点编辑的新技术。由于它具有设计简单、操作方便、费用低廉等巨大优势,给遗传操作领域带来了一场革命性的改变。本文重点介绍了CRISPR/Cas9系统在基因组DNA片段靶向编辑方面的研究和应用,主要包括DNA片段的删除、反转、重复、插入和易位,这一有效的DNA片段编辑方法为研究基因功能、调控元件、组织发育和疾病发生发展提供了有力手段。本文最后展望了Ⅱ型CRISPR/Cas9系统的应用前景和其他类型CRISPR系统的应用潜力,为开展利用基因组DNA片段靶向编辑进行基因调控和功能研究提供参考。     

RAD-seq技术在基因组研究中的现状及展望

Restriction-site associated DNA sequencing(RAD-seq)技术是在二代测序基础上发展起来的一项基于全基因组酶切位点的简化基因组测序技术。该方法技术流程简单, 不受有无参考基因组的限制, 可大大简化基因组的复杂性, 减少实验费用, 通过一次测序就可以获得数以万计的多态性标记。目前, RAD-seq技术已成功应用于超高密度遗传图谱的构建、重要性状的精细定位、辅助基因组序列组装、群体基因组学以及系统发生学等基因组研究热点领域。文章主要介绍了RAD-seq的技术原理、技术发展及其在基因组研究中的广泛应用。鉴于RAD-seq方法的独特性, 该技术必将在复杂基因组研究领域具有广泛的应用前景。     

A method for site-directed transplacement of in vitro altered DNA sequences in Rhizobium

Summary A method that directs transplacement of in vitro altered DNA sequences to substitute the corresponding wild-type DNA sequences at the original location in the Rhizobium genome is described. The NPTII gene of transposon Tn5 which confers resistance to several antibiotics in a wide variety of organisms is used as a selectable marker on the vector. To generate DNA fragment substitution, it is not essential to place a selectable genetic marker directly linked to the in vitro altered region of the DNA fragment. The method utilizes the vector pSUP201 that replicates in E. coli but neither in Rhizobium nor in many other host systems. DNA transplacement occurs by the integration of the in vitro altered DNA fragment at its homologous site in the chromosome along with the vector. A duplicated target sequence is generated, which is then followed by a homologous recombination event between the duplicated DNA sequences leading to the excision of the vector carrying the selectable marker together with one of the duplicated copies of DNA. The excised DNA fragment is subsequently lost due to the inability of the vector pSUP201 to replicate in Rhizobium. Using this method, we have transplaced a nif DNA fragment containing a large deletion of approximately 9 Kb into its homologous site in the genome of cowpea Rhizobium IRc78. The IRc78 strain carrying the deletion forms nodules on host-plants but is unable to reduce atmospheric nitrogen. The method allows precise alterations of the genomic DNA in an organism that is genetically not well characterized, provided a vector carrying a selectable marker can be transferred.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Preparation of a genomic library using a TA vector

An efficient and simple method for constructing a genomic DNA library is presented using a TA cloning vector. It is based on sonication cleavage of genomic DNA, blunting of the fragment ends with mung bean nuclease, and addition of a single 3'-deoxyadenylate with Taq DNA polymerase, followed by ligation with a TA vector. This method is useful for improving the quality of genomic libraries for organisms whose genomic DNA is not well digested with restriction enzymes owing to the presence of polysaccharides and/or DNA methylation.     

Optimum conditions for selective isolation of genes from complex genomes by transformation-associated recombination cloning

Transformation-associated recombination (TAR) cloning in yeast is used to isolate a desired chromosomal region or gene from a complex genome without construction of a genomic library. The technique involves homologous recombination during yeast spheroplast transformation between genomic DNA and a TAR vector containing short 5′ and 3′ gene-specific targeting hooks. Efficient gene capture requires a high yield of transformants, and we demonstrate here that the transformant yield increases ~10-fold when the genomic DNA is sheared to 100–200 kb before being presented to the spheroplasts. Here we determine the most effective concentration of genomic DNA, and also show that the targeted sequences recombine much more efficiently with the vector’s targeting hooks when they are located at the ends of the genomic DNA fragment. We demonstrate that the yield of gene-positive clones increases ~20-fold after endonuclease digestion of genomic DNA, which caused double strand breaks near the targeted sequences. These findings have led to a greatly improved protocol.     

CRISPR/Cas9 and Genome Editing in Drosophila

Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively easily. Traditional techniques for generating genetic mutations in most organisms have relied on selection from large pools of randomly induced mutations for those of particular interest, or time-consuming gene targeting by homologous recombination. Drosophila melanogaster has always been at the forefront of genetic analysis, and application of these new genome editing techniques to this organism will revolutionise our approach to performing analysis of gene function in the future. We discuss the recent techniques that apply the CRISPR/Cas9 system to Drosophila, highlight potential uses for this technology and speculate upon the future of genome engineering in this model organism.