Genome Sequencer FLX引领快速基因组测序时代的到来

焦磷酸法最早是应用在检测DNA甲基化和SNP位点分析的一项技术, 在2005年底, 此技术被用来进行基因组序列的测定, 已经成为下一代高通量测序中最成熟的一种技术。本篇文章着重介绍了采用焦磷酸测序法的罗氏公司最新一代高通量基因组测序系统GS FLX的技术原理, 操作过程和广泛的应用范围。     

一种快速高效的全基因组甲基化CpG岛富集方法的建立

高甲基化的CpG岛所致基因表观遗传学转录失活已经成为肿瘤表观基因组学研究的重要内容。现在已有很多检测CpG岛甲基化的方法,但由于各自的局限,还没有建立一种能快速在全基因组水平上进行甲基化CpG岛的富集方法。本研究利用甲基化结合蛋白MBD2b具有特异性结合甲基化DNA的特性, 建立了一种基于DNA免疫共沉淀技术的全基因组甲基化CpG岛的富集方法。在大肠杆菌中表达重组的GST-MBD2b蛋白,通过Glutathione Sepharose 4B对重组蛋白进行纯化,制备成亲和层析柱,利用在不同的盐离子强度下甲基化DNA和非甲基化DNA的结合能力不同,对甲基化DNA进行富集。用甲基化酶SssI处理过的DNA片段与非甲基化DNA片段进行富集效率的检测,发现0.5M KCl的浓度是甲基化DNA片段和非甲基化DNA片段得以分开的临界条件。样品的富集效率用Real Time PCR进行检测。结果表明,这种方法能够实现对全基因组甲基化DNA的有效富集且最高的富集倍数可达到100多倍。富集到的甲基化DNA可以进行后续的定量PCR, DNA测序和全基因组芯片的分析等工作,为大规模分析全基因组CpG 岛甲基化的改变奠定了基础。     

Impact of Next Generation Sequencing Techniques in Food Microbiology

Understanding the Maxam-Gilbert and Sanger sequencing as the first generation, in recent years there has been an explosion of newly-developed sequencing strategies, which are usually referred to as next generation sequencing (NGS) techniques. NGS techniques have high-throughputs and produce thousands or even millions of sequences at the same time. These sequences allow for the accurate identification of microbial taxa, including uncultivable organisms and those present in small numbers. In specific applications, NGS provides a complete inventory of all microbial operons and genes present or being expressed under different study conditions. NGS techniques are revolutionizing the field of microbial ecology and have recently been used to examine several food ecosystems. After a short introduction to the most common NGS systems and platforms, this review addresses how NGS techniques have been employed in the study of food microbiota and food fermentations, and discusses their limits and perspectives. The most important findings are reviewed, including those made in the study of the microbiota of milk, fermented dairy products, and plant-, meat- and fish-derived fermented foods. The knowledge that can be gained on microbial diversity, population structure and population dynamics via the use of these technologies could be vital in improving the monitoring and manipulation of foods and fermented food products. They should also improve their safety.     

A large‐scale gene discovery for the red palm weevil Rhynchophorus ferrugineus (Coleoptera: Curculionidae)

The red palm weevil (RPW; Rhynchophorus ferrugineus) is a devastating pest of palms, prevalent in the Middle East as well as many other regions of the world. Here, we report a large‐scale de novo complementary DNA (cDNA) sequencing effort that acquired ～5 million reads and assembled them into 26 765 contigs from 12 libraries made from samples of different RPW developmental stages based on the Roche/454 GS FLX platform. We annotated these contigs based on the publically available known insect genes and the Tribolium castaneum genome assembly. We find that over 80% of coding sequences (CDS) from the RPW contigs have high‐identity homologs to known proteins with complete CDS. Gene expression analysis shows that the pupa and larval stages have the highest and lowest expression levels, respectively. In addition, we also identified more than 60 000 single nucleotide polymorphisms and 1 200 simple sequence repeat markers. This study provides the first large‐scale cDNA dataset for RPW, a much‐needed resource for future molecular studies.     

二代及三代测序技术在遗传学诊断中的应用进展

遗传病的防治是公共卫生领域的重大课题,而明确病因是遗传病防治的重要环节。高通量测序技术（又称二代测序技术）具有高通量、低成本、高准确度的优点,为遗传诊断及咨询提供了直接证据,已成为遗传学检测不可或缺的有力工具;第三代测序也凭借其长读长的独特优势在临床应用中占据一席之地。二代及三代测序技术各有特点,互为补充,临床中针对不同的检测需求有多种类型的测序方案可供选择。基于此,对二代及三代测序技术的原理、分类及其在遗传学诊断中的应用进展做一综述,以期为临床测序方案的选择提供思路和指导。     

Comparison of random and SSR-enriched shotgun pyrosequencing for microsatellite discovery and single multiplex PCR optimization in Acacia harpophylla F. Muell. Ex Benth

Streamlining the development and genotyping of microsatellites in species for which no genetic information is available represents an important technical challenge to overcome in order to enable mainstream application of state-of-the-art population genetic analysis techniques in nonmodel organisms. Using the example of Acacia harpophylla, an acacia tree endemic of north-eastern Australia, we show that high-throughput shotgun pyrosequencing technology, so-called second-generation sequencing, reduces time and cost of microsatellite marker discovery in nonmodel organisms and of their large-scale typing in natural populations. We found that 0.5% of short sequence reads generated on 454 Genome Sequencer FLX Titanium from random genome sampling and 2.2% of reads generated with prior microsatellite enrichment yielded microsatellite markers with designed polymerase chain reaction (PCR) primers, suggesting that enrichment increases efficiency of pyrosequencing when microsatellite discovery is the primary goal. Using stringent selection criteria to facilitate downstream PCR multiplex design, we identified 1435 microsatellite loci with designed primers from a total of 200,908 short sequence reads. From a subset of 96 loci tested for amplification, 38 were validated for population genetics applications, leading to the optimization of a cost-effective multiplex PCR protocol for the simultaneous typing of nine microsatellites in natural populations of A. harpophylla.     

GS6, A Member of the GRAS Gene Family,Negatively Regulates Grain Size in Rice

Grain size is an important yield-related trait in rice. Intensive artificial selection for grain size during domestication is evidenced by the larger grains of most of today’s cultivars compared with t...     

High-performance liquid chromatographic determination of GS4071, a potent inhibitor of influenza neuraminidase, in plasma by precolumn fluorescence derivatization with naphthalenedialdehyde

GS4071 is a potent inhibitor of influenza neuraminidase. A precolumn fluorescence derivatization HPLC method is described for the analysis of GS4071 in rat plasma. Plasma samples were subjected to solid-phase extraction on C₁₈ extraction columns. After extraction, GS4071 was derivatized with naphthalenedialdehyde in the presence of potassium cyanide to produce highly fluorescent cyano[f]benzoisoindole derivatives. Derivatized samples were stable for >24 h at 4°C. The samples were analyzed by an isocratic HPLC method using fluorescence detection at 420 nm excitation and 470 nm emission wavelength. The method was validated and applied to the analysis of plasma samples from pre-clinical pharmacokinetic studies in rats. The limit of detection for GS4071 was 20 ng/ml. For five replicate samples at 50, 400, and 1000 ng/ml, the within-day precision values were 16.9, 9.4 and 4.5%, respectively, and the between-day precision values were 16.9, 7.9, and 2.1%, respectively. The method was linear from 25 to 1600 ng/ml and the total recovery was >68% over this concentration range.     

Identification of Translin and Trax as Components of the GS1 Strand-Specific DNA Binding Complex Enriched in Brain

Abstract: In previous gel-shift assays, we identified a protein complex, referred to as GS1, that binds in a sequence-specific manner to single-stranded DNA and is highly enriched in brain. As an initial step in clarifying the function of this complex, we have undertaken studies aimed at defining its protein components. In particular, we focused on identifying two protein bands that were covalently labeled when the GS1-DNA complex was subjected to UV irradiation to induce cross-linking between the radiolabeled probe and GS1 components. By following GS1 binding activity through a series of conventional chromatographic steps, as well as an affinity column based on the DNA oligonucleotide used for gel-shift assays, we were able to achieve ∼500,000-fold enrichment of GS1 compared with that in crude cerebellar extracts used as starting material. This highly purified fraction contained both protein bands detected by UV cross-linking in crude extracts. Sequencing of peptides derived from these proteins led to their identification as Translin and Trax, interacting proteins identified in studies of DNA recombination in lymphocytes. A distinct line of research has provided evidence that a complex containing Translin can bind to specific mRNAs and block their translation. Whether one or both of these proposed functions of the Translin/Trax complex explains the high basal level of GS1 binding activity present in the brain remains to be determined.     

HCV特异性M1GS核酶的构建及体外切割活性研究

针对HCV基因组中较为保守的区域-5'UTR,设计一段GS引导序列,并与大肠杆菌RNase P的催化亚基-M1RNA的3'末端共价结合,构建序列特异性M1GS核酶-M1GS-HCV/C20。体外实验证实,所构建的人工核酶对HCV 5'UTR具有明显的靶向切割活性,且这种切割发生于靶序列的特定位点。本研究将为进一步阐明该核酶在胞内的活性、乃至动物模型内评价其抗病毒效果提供实验材料,从而为新型抗HCV药物及反义基因治疗的研究奠定基础。     

Wnt信号通路GS蛋白在胃癌中的表达和临床意义

目的：研究wnt信号通路的中GS蛋白（谷氨酰胺合成酶）在胃癌组织中的表达,探索其在胃癌发生、发展中的意义。方法：用免疫组织化学法测定胃癌组织（110例）、肠化生组织（30例）、不典型增生组织（20例）及慢性浅表性胃炎组织（60例）中GS蛋白表达。用快速尿素酶法与病理组织切片染色法检测上述各组织中HP感染的情况,并予统计学分析比较其差异。结果：胃癌组织GS高蛋白表达与组织分型、分化程度、淋巴结转移密切相关（P〈0．05）,与肿瘤大小、部位、TNM分期、Borrmann分型、性别、年龄等无明显相关（P〉0．05）。GS表达与HP感染密切相关。结论：GS蛋白高表达同胃癌生物学行为密切相关,在胃癌的发生、发展中起重要作用。